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Toone, Eric John

Overview:

Dr. Toone is a physical organic chemist who studies relationships between structure and activity in the context of biology. Currently active programs exist in biocatalysis/applied enzymology, ligand binding and the activity of water, and the synthesis of novel donors of nitric oxide. The study of these problems makes use of synthetic organic chemistry,
traditional enzymology, isothermal titration
microcalorimetry, and the techniques of directed evolution.

Positions:

Professor of Chemistry

Chemistry
Trinity College of Arts & Sciences

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Faculty Network Member of The Energy Initiative

Duke University Energy Initiative
Institutes and Provost's Academic Units

Professor of Biochemistry

Biochemistry
School of Medicine

Bass Fellow

Chemistry
Trinity College of Arts & Sciences

Education:

B.S. 1983

B.S. — University of Guelph (Canada)

M.S. 1985

M.S. — University of Toronto (Canada)

Ph.D. 1988

Ph.D. — University of Toronto (Canada)

News:

Grants:

Triangle Venture Alliance

Administered By
Duke Innovation & Entrepreneurship
AwardedBy
NC IDEA
Role
Principal Investigator
Start Date
March 01, 2017
End Date
June 30, 2019

Adjuvant Discovery Program (Option #1)

Administered By
Pathology
AwardedBy
National Institutes of Health
Role
Project Leader
Start Date
September 30, 2014
End Date
September 29, 2017

Adjuvant Discovery Program (Option #1)

Administered By
Pathology
AwardedBy
National Institutes of Health
Role
Project Leader
Start Date
September 30, 2014
End Date
September 29, 2017

Adjuvant Discovery Program

Administered By
Pathology
AwardedBy
National Institutes of Health
Role
Project Leader
Start Date
September 30, 2014
End Date
April 04, 2017

Adjuvant Discovery Program

Administered By
Pathology
AwardedBy
National Institutes of Health
Role
Project Leader
Start Date
September 30, 2014
End Date
April 04, 2017

LpxC inhibitors as a novel class of antibiotics against N. gonorrhoeae

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
April 01, 2011
End Date
March 31, 2017

LpxC inhibitors as a novel class of antibiotics against N. gonorrhoeae

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
April 01, 2011
End Date
March 31, 2017

Structural and Biochemical Studies of LpxC Inhibition

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
June 15, 2003
End Date
March 31, 2014

Structural and Biochemical Studies of LpxC Inhibition

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
June 15, 2003
End Date
March 31, 2014

Replacement Equipment Components for an 800 MHz NMR Spectrometer

Administered By
Radiology
AwardedBy
National Institutes of Health
Role
Major User
Start Date
August 20, 2010
End Date
August 19, 2011

Replacement Equipment Components for an 800 MHz NMR Spectrometer

Administered By
Radiology
AwardedBy
National Institutes of Health
Role
Major User
Start Date
August 20, 2010
End Date
August 19, 2011

Purchase of a Q-TOF LC/MS System

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Major User
Start Date
August 09, 2010
End Date
August 08, 2011

Purchase of a Q-TOF LC/MS System

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Major User
Start Date
August 09, 2010
End Date
August 08, 2011

NIRT: Hierarchical Bionanomanufacturing

Administered By
Pratt School of Engineering
AwardedBy
National Science Foundation
Role
Co-Principal Investigator
Start Date
September 15, 2006
End Date
August 31, 2010

NIRT: Hierarchical Bionanomanufacturing

Administered By
Pratt School of Engineering
AwardedBy
National Science Foundation
Role
Co-Principal Investigator
Start Date
September 15, 2006
End Date
August 31, 2010

NIRT: Hierarchical Bionanomanufacturing

Administered By
Pratt School of Engineering
AwardedBy
National Science Foundation
Role
Co-Principal Investigator
Start Date
September 15, 2006
End Date
August 31, 2010

Biocatalytic Nanolithography

Administered By
Pratt School of Engineering
AwardedBy
National Science Foundation
Role
Co-Principal Investigator
Start Date
July 01, 2006
End Date
June 30, 2010

Biocatalytic Nanolithography

Administered By
Pratt School of Engineering
AwardedBy
National Science Foundation
Role
Co-Principal Investigator
Start Date
July 01, 2006
End Date
June 30, 2010

Biocatalytic Nanolithography

Administered By
Pratt School of Engineering
AwardedBy
National Science Foundation
Role
Co-Principal Investigator
Start Date
July 01, 2006
End Date
June 30, 2010

Tissue Transglutaminase in Neurologic Injury

Administered By
Medicine, Hematology
AwardedBy
National Institutes of Health
Role
Investigator
Start Date
July 01, 2006
End Date
June 30, 2009

Tissue Transglutaminase in Neurologic Injury

Administered By
Medicine, Hematology
AwardedBy
National Institutes of Health
Role
Investigator
Start Date
July 01, 2006
End Date
June 30, 2009

The Evolution of Synthetically Useful Aldolases

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 2002
End Date
December 31, 2006

The Evolution of Synthetically Useful Aldolases

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 2002
End Date
December 31, 2006

Origin and Application of the Cluster Glycoside Effect

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 1999
End Date
December 31, 2006

Origin and Application of the Cluster Glycoside Effect

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 1999
End Date
December 31, 2006

Structure and Regulation of hsp90 Chaperones

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
May 01, 2005
End Date
October 31, 2005

Structure and Regulation of hsp90 Chaperones

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
May 01, 2005
End Date
October 31, 2005

(93-0250) Pyruvate Aldolases in Organic Synthesis

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1993
End Date
June 30, 1998

(93-0250) Pyruvate Aldolases in Organic Synthesis

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1993
End Date
June 30, 1998

(94-0802) Pyruvate Aldolases in Organic Synthesis

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1993
End Date
June 30, 1998

(94-0802) Pyruvate Aldolases in Organic Synthesis

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1993
End Date
June 30, 1998

(95-0831) Pyruvate Aldolases in Organic Synthesis

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1993
End Date
June 30, 1998

(95-0831) Pyruvate Aldolases in Organic Synthesis

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1993
End Date
June 30, 1998

(96-0886) Pyruvate Aldolases in Organic Synthesis

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1993
End Date
June 30, 1998

(96-0886) Pyruvate Aldolases in Organic Synthesis

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1993
End Date
June 30, 1998

(97-0817) Pyruvate Aldolases in Organic Synthesis

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1993
End Date
June 30, 1998

(97-0817) Pyruvate Aldolases in Organic Synthesis

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1993
End Date
June 30, 1998

(95-0434) Structure and Energetics of Protein-Carbohydrate Complexes

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1994
End Date
September 30, 1996

(95-0434) Structure and Energetics of Protein-Carbohydrate Complexes

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1994
End Date
September 30, 1996

(93-0249) Structure and Energetics of Protein-carbohydrate Complexes

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1994
End Date
March 31, 1996

(93-0249) Structure and Energetics of Protein-carbohydrate Complexes

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1994
End Date
March 31, 1996
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Awards:

Sloan Research Fellowship-Chemistry. Alfred P. Sloan Foundation.

Type
National
Awarded By
Alfred P. Sloan Foundation
Date
January 01, 1995

Publications:

Specific binding at the cellulose binding module-cellulose interface observed by force spectroscopy.

The need for effective enzymatic depolymerization of cellulose has stimulated an interest in interactions between protein and cellulose. Techniques utilized for quantitative measurements of protein-cellulose noncovalent association include microgravimetry, calorimetry, and atomic force microscopy (AFM), none of which differentiate between specific protein-cellulose binding and nonspecific adhesion. Here, we describe an AFM approach that differentiates nonspecific from specific interactions between cellulose-binding modules (CBMs) and cellulose. We demonstrate that the "mismatched" interaction between murine galectin-3, a lectin with no known affinity for cellulose, and cellulose shows molecular recognition force microscopy profiles similar to those observed during the interaction of a "matched" clostridial CBM3a with the same substrate. We also examine differences in binding probabilities and rupture profiles during CBM-cellulose binding experiments in the presence and absence of a blocking agent-a substrate specific for CBM that presumably blocks binding sites. By comparison of the behavior of the two proteins, we separate specific (i.e., blockable) and nonspecific adhesion events and show that both classes of interaction exhibit nearly identical rupture forces (45 pN at ∼0.4 nN/s). Our work provides an important caveat for the interpretation of protein-carbohydrate binding by force spectroscopy; delineation of the importance of such interactions to other classes of binding warrants further study.

Authors
King, JR; Bowers, CM; Toone, EJ
MLA Citation
King, JR, Bowers, CM, and Toone, EJ. "Specific binding at the cellulose binding module-cellulose interface observed by force spectroscopy." Langmuir : the ACS journal of surfaces and colloids 31.11 (March 12, 2015): 3431-3440.
PMID
25738531
Source
epmc
Published In
Langmuir
Volume
31
Issue
11
Publish Date
2015
Start Page
3431
End Page
3440
DOI
10.1021/la504836u

Diamond surface functionalization with biomimicry - Amine surface tether and thiol moiety for electrochemical sensors

The surface of conducting diamond was functionalized with a terminal thiol group that is capable of binding and detecting nitrogen-oxygen species. The functionalization process employed multiple steps starting with doped diamond films grown by plasma enhanced chemical vapor deposition followed by hydrogen termination and photochemical attachment of a chemically protected amine alkene. The surface tether was deprotected to reveal the amine functionality, which enabled the tether to be extended with surface chemistry to add a terminal thiol moiety for electrochemical sensing applications. Each step of the process was validated using X-ray photoelectron spectroscopy analysis. © 2014 Elsevier B.V. All rights reserved.

Authors
Sund, JB; Causey, CP; Wolter, SD; Parker, CB; Stoner, BR; Toone, EJ; Glass, JT
MLA Citation
Sund, JB, Causey, CP, Wolter, SD, Parker, CB, Stoner, BR, Toone, EJ, and Glass, JT. "Diamond surface functionalization with biomimicry - Amine surface tether and thiol moiety for electrochemical sensors." Applied Surface Science 301 (May 15, 2014): 293-299.
Source
scopus
Published In
Applied Surface Science
Volume
301
Publish Date
2014
Start Page
293
End Page
299
DOI
10.1016/j.apsusc.2014.02.067

Diamond surface functionalization with biomimicry – Amine surface tether and thiol moiety for electrochemical sensors

Authors
Sund, JB; Causey, CP; Wolter, SD; Parker, CB; Stoner, BR; Toone, EJ; Glass, JT
MLA Citation
Sund, JB, Causey, CP, Wolter, SD, Parker, CB, Stoner, BR, Toone, EJ, and Glass, JT. "Diamond surface functionalization with biomimicry – Amine surface tether and thiol moiety for electrochemical sensors." Applied Surface Science 301 (May 2014): 293-299.
Source
crossref
Published In
Applied Surface Science
Volume
301
Publish Date
2014
Start Page
293
End Page
299
DOI
10.1016/j.apsusc.2014.02.067

Structural Modifications in Bilayered Molecular Systems Lead to Predictable Changes in Their Electronic Properties

Authors
Bowers, CM; Zhang, M; Lyubarskaya, Y; Toone, EJ; Tang, C; Shestopalov, AA
MLA Citation
Bowers, CM, Zhang, M, Lyubarskaya, Y, Toone, EJ, Tang, C, and Shestopalov, AA. "Structural Modifications in Bilayered Molecular Systems Lead to Predictable Changes in Their Electronic Properties." Advanced Materials Interfaces 1.2 (April 2014): 1300109-1300109.
Source
crossref
Published In
Advanced Materials Interfaces
Volume
1
Issue
2
Publish Date
2014
Start Page
1300109
End Page
1300109
DOI
10.1002/admi.201300109

Structural basis of the promiscuous inhibitor susceptibility of Escherichia coli LpxC.

The LpxC enzyme in the lipid A biosynthetic pathway is one of the most promising and clinically unexploited antibiotic targets for treatment of multidrug-resistant Gram-negative infections. Progress in medicinal chemistry has led to the discovery of potent LpxC inhibitors with a variety of chemical scaffolds and distinct antibiotic profiles. The vast majority of these compounds, including the nanomolar inhibitors L-161,240 and BB-78485, are highly effective in suppressing the activity of Escherichia coli LpxC (EcLpxC) but not divergent orthologs such as Pseudomonas aeruginosa LpxC (PaLpxC) in vitro. The molecular basis for such promiscuous inhibition of EcLpxC has remained poorly understood. Here, we report the crystal structure of EcLpxC bound to L-161,240, providing the first molecular insight into L-161,240 inhibition. Additionally, structural analysis of the EcLpxC/L-161,240 complex together with the EcLpxC/BB-78485 complex reveals an unexpected backbone flipping of the Insert I βa-βb loop in EcLpxC in comparison with previously reported crystal structures of EcLpxC complexes with l-threonyl-hydroxamate-based broad-spectrum inhibitors. Such a conformational switch, which has only been observed in EcLpxC but not in divergent orthologs such as PaLpxC, results in expansion of the active site of EcLpxC, enabling it to accommodate LpxC inhibitors with a variety of head groups, including compounds containing single (R- or S-enantiomers) or double substitutions at the neighboring Cα atom of the hydroxamate warhead group. These results highlight the importance of understanding inherent conformational plasticity of target proteins in lead optimization.

Authors
Lee, C-J; Liang, X; Gopalaswamy, R; Najeeb, J; Ark, ED; Toone, EJ; Zhou, P
MLA Citation
Lee, C-J, Liang, X, Gopalaswamy, R, Najeeb, J, Ark, ED, Toone, EJ, and Zhou, P. "Structural basis of the promiscuous inhibitor susceptibility of Escherichia coli LpxC." ACS Chem Biol 9.1 (January 17, 2014): 237-246.
PMID
24117400
Source
pubmed
Published In
ACS Chemical Biology
Volume
9
Issue
1
Publish Date
2014
Start Page
237
End Page
246
DOI
10.1021/cb400067g

Photo-activated psoralen binds the ErbB2 catalytic kinase domain, blocking ErbB2 signaling and triggering tumor cell apoptosis.

Photo-activation of psoralen with UVA irradiation, referred to as PUVA, is used in the treatment of proliferative skin disorders. The anti-proliferative effects of PUVA have been largely attributed to psoralen intercalation of DNA, which upon UV treatment, triggers the formation of interstrand DNA crosslinks (ICL) that inhibit transcription and DNA replication. Here, we show that PUVA exerts antitumor effects in models of human breast cancer that overexpress the ErbB2 receptor tyrosine kinase oncogene, through a new mechanism. Independent of ICL formation, the antitumor effects of PUVA in ErbB2+ breast cancer models can instead be mediated through inhibition of ErbB2 activation and signaling. Using a mass spectroscopy-based approach, we show for the first time that photo-activated 8MOP (8-methoxypsoralen) interacts with the ErbB2 catalytic autokinase domain. Furthermore, PUVA can reverse therapeutic resistance to lapatinib and other ErbB2 targeted therapies, including resistance mediated via expression of a phosphorylated, truncated form of ErbB2 (p85(ErbB2)) that is preferentially expressed in tumor cell nuclei. Current ErbB2 targeted therapies, small molecule kinase inhibitors or antibodies, do not block the phosphorylated, activated state of p85(ErbB2). Here we show that PUVA reduced p85(ErbB2) phosphorylation leading to tumor cell apoptosis. Thus, in addition to its effects on DNA and the formation of ICL, PUVA represents a novel ErbB2 targeted therapy for the treatment of ErbB2+ breast cancers, including those that have developed resistance to other ErbB2 targeted therapies.

Authors
Xia, W; Gooden, D; Liu, L; Zhao, S; Soderblom, EJ; Toone, EJ; Beyer, WF; Walder, H; Spector, NL
MLA Citation
Xia, W, Gooden, D, Liu, L, Zhao, S, Soderblom, EJ, Toone, EJ, Beyer, WF, Walder, H, and Spector, NL. "Photo-activated psoralen binds the ErbB2 catalytic kinase domain, blocking ErbB2 signaling and triggering tumor cell apoptosis." PloS one 9.2 (January 2014): e88983-.
PMID
24551203
Source
epmc
Published In
PloS one
Volume
9
Issue
2
Publish Date
2014
Start Page
e88983
DOI
10.1371/journal.pone.0088983

Structural Basis of the Promiscuous Inhibitor Susceptibility of Escherichia coli LpxC

Authors
Lee, C-J; Liang, X; Gopalaswamy, R; Najeeb, J; Ark, ED; Toone, EJ; Zhou, P
MLA Citation
Lee, C-J, Liang, X, Gopalaswamy, R, Najeeb, J, Ark, ED, Toone, EJ, and Zhou, P. "Structural Basis of the Promiscuous Inhibitor Susceptibility of Escherichia coli LpxC." ACS CHEMICAL BIOLOGY 9.1 (January 2014): 237-246.
Source
wos-lite
Published In
ACS Chemical Biology
Volume
9
Issue
1
Publish Date
2014
Start Page
237
End Page
246
DOI
10.1021/cb400067g

Erratum: Eenthalpic signature of methonium desolvation revealed in a synthetic host-guest system based on cucurbit[7]uril (Journal of the American Chemical Society (2013) 135 (6084-6091) DOI: 10.1021/ja311327v)

Authors
Wang, Y; King, JR; Wu, P; Pelzman, DL; Beratan, DN; Toone, EJ
MLA Citation
Wang, Y, King, JR, Wu, P, Pelzman, DL, Beratan, DN, and Toone, EJ. "Erratum: Eenthalpic signature of methonium desolvation revealed in a synthetic host-guest system based on cucurbit[7]uril (Journal of the American Chemical Society (2013) 135 (6084-6091) DOI: 10.1021/ja311327v)." Journal of the American Chemical Society 135.46 (November 20, 2013): 17650-.
Source
scopus
Published In
Journal of the American Chemical Society
Volume
135
Issue
46
Publish Date
2013
Start Page
17650
DOI
10.1021/ja408346j

Correction to "Enthalpic Signature of Methonium Desolvation Revealed in a Synthetic Host-Guest System Based on Cucurbit[7]uril".

Authors
Wang, Y; King, JR; Wu, P; Pelzman, DL; Beratan, DN; Toone, EJ
MLA Citation
Wang, Y, King, JR, Wu, P, Pelzman, DL, Beratan, DN, and Toone, EJ. "Correction to "Enthalpic Signature of Methonium Desolvation Revealed in a Synthetic Host-Guest System Based on Cucurbit[7]uril"." J Am Chem Soc 135.46 (November 20, 2013): 17650-.
PMID
24215303
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
135
Issue
46
Publish Date
2013
Start Page
17650
DOI
10.1021/ja408346j

Synthesis, structure, and antibiotic activity of aryl-substituted LpxC inhibitors.

The zinc-dependent deacetylase LpxC catalyzes the committed step of lipid A biosynthesis in Gram-negative bacteria and is a validated target for the development of novel antibiotics to combat multidrug-resistant Gram-negative infections. Many potent LpxC inhibitors contain an essential threonyl-hydroxamate headgroup for high-affinity interaction with LpxC. We report the synthesis, antibiotic activity, and structural and enzymatic characterization of novel LpxC inhibitors containing an additional aryl group in the threonyl-hydroxamate moiety, which expands the inhibitor-binding surface in LpxC. These compounds display enhanced potency against LpxC in enzymatic assays and superior antibiotic activity against Francisella novicida in cell culture. The comparison of the antibiotic activities of these compounds against a leaky Escherichia coli strain and the wild-type strain reveals the contribution of the formidable outer-membrane permeability barrier that reduces the compounds efficacy in cell culture and emphasizes the importance of maintaining a balanced hydrophobicity and hydrophilicity profile in developing effective LpxC-targeting antibiotics.

Authors
Liang, X; Lee, C-J; Zhao, J; Toone, EJ; Zhou, P
MLA Citation
Liang, X, Lee, C-J, Zhao, J, Toone, EJ, and Zhou, P. "Synthesis, structure, and antibiotic activity of aryl-substituted LpxC inhibitors." J Med Chem 56.17 (September 12, 2013): 6954-6966.
PMID
23914798
Source
pubmed
Published In
Journal of Medicinal Chemistry
Volume
56
Issue
17
Publish Date
2013
Start Page
6954
End Page
6966
DOI
10.1021/jm4007774

The 62nd Annual Meeting of the Congress of Neurological Surgeons was held in Chicago, Illinois from October 6-10, 2012. Preface.

Authors
Grant, GA; Hankinson, T; Muh, C; Dumont, A
MLA Citation
Grant, GA, Hankinson, T, Muh, C, and Dumont, A. "The 62nd Annual Meeting of the Congress of Neurological Surgeons was held in Chicago, Illinois from October 6-10, 2012. Preface." Neurosurgery 60 Suppl 1 (August 2013): v-.
PMID
23839472
Source
epmc
Published In
Neurosurgery
Volume
60 Suppl 1
Publish Date
2013
Start Page
v
DOI
10.1227/neu.0000000000000006

Effect of compressive force on unbinding specific protein-ligand complexes with force spectroscopy.

Atomic force microscopy (AFM) is used extensively for the investigation of noncovalent molecular association. Although the technique is used to derive various types of information, in almost all instances the frequency of complex formation, the magnitude of rupture forces, and the shape of the force-distance curve are used to determine the behavior of the system. We have used AFM to consider the effect of contact force on the unbinding profiles of lactose-galectin-3, as well as the control pairs lactose-KDPG aldolase, and mannose-galectin-3, where the interacting species show negligible solution-phase affinity. Increased contact forces (>250 pN) resulted in increased probabilitites of binding and decreased blocking efficiencies for the cognate ligand-receptor pair lactose-G3. Increased contact force applied to two control systems with no known affinity, mannose-G3 and lactose-KDPG aldolase, resulted in nonspecific ruptures that were indistinguishable from those of specific lactose-G3 interactions. These results demonstrate that careful experimental design is vital to the production of interpretable data, and suggest that contact force minimization is an effective technique for probing the unbinding forces and rupture lengths of only specific ligand-receptor interactions.

Authors
Bowers, CM; Carlson, DA; Rivera, M; Clark, RL; Toone, EJ
MLA Citation
Bowers, CM, Carlson, DA, Rivera, M, Clark, RL, and Toone, EJ. "Effect of compressive force on unbinding specific protein-ligand complexes with force spectroscopy." J Phys Chem B 117.17 (May 2, 2013): 4755-4762.
PMID
23537272
Source
pubmed
Published In
The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces and Biophysical
Volume
117
Issue
17
Publish Date
2013
Start Page
4755
End Page
4762
DOI
10.1021/jp309393s

Enthalpic signature of methonium desolvation revealed in a synthetic host-guest system based on cucurbit[7]uril.

Methonium (N(+)Me3) is an organic cation widely distributed in biological systems. As an organic cation, the binding of methonium to protein receptors requires the removal of a positive charge from water. The appearance of methonium in biological transmitters and receptors seems at odds with the large unfavorable desolvation free energy reported for tetramethylammonium (TMA(+)), a frequently utilized surrogate of methonium. Here, we report an experimental system that facilitates incremental internalization of methonium within the molecular cavity of cucurbit[7]uril (CB[7]). Using a combination of experimental and computational studies, we show that the transfer of methonium from bulk water (partially solvated methonium state) to the CB[7] cavity (mostly desolvated methonium state) is accompanied by a remarkably small desolvation enthalpy of just 0.5 ± 0.3 kcal·mol(-1), a value significantly less endothermic than those values suggested from gas-phase model studies. Our results are in accord with neutron scattering measurements that suggest methonium produces only a minimal perturbation in the bulk water structure, which highlights the limitations of gas-phase models. More surprisingly, the incremental withdrawal of the methonium surface from water produces a nonmonotonic response in desolvation enthalpy. A partially desolvated state exists, in which a portion of the methonium group remains exposed to solvent. This structure incurs an increased enthalpic penalty of ~3 kcal·mol(-1) compared to other solvation states. We attribute this observation to the pre-encapsulation dewetting of the methonium surface. Together, our results offer a rationale for the wide distribution of methonium in a biological context and suggest limitations to computational estimates of binding affinities based on simple parametrization of solvent-accessible surface area.

Authors
Wang, Y; King, JR; Wu, P; Pelzman, DL; Beratan, DN; Toone, EJ
MLA Citation
Wang, Y, King, JR, Wu, P, Pelzman, DL, Beratan, DN, and Toone, EJ. "Enthalpic signature of methonium desolvation revealed in a synthetic host-guest system based on cucurbit[7]uril." J Am Chem Soc 135.16 (April 24, 2013): 6084-6091.
PMID
23510488
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
135
Issue
16
Publish Date
2013
Start Page
6084
End Page
6091
DOI
10.1021/ja311327v

Energy biotechnology in 2013: advanced technology development for breakthroughs in fuels and chemicals production

Authors
Toone, E; Winde, HD
MLA Citation
Toone, E, and Winde, HD. "Energy biotechnology in 2013: advanced technology development for breakthroughs in fuels and chemicals production." Current Opinion in Biotechnology (2013).
PMID
23692669
Source
scival
Published In
Current Opinion in Biotechnology
Publish Date
2013
DOI
10.1016/j.copbio.2013.04.009

Energy biotechnology in 2013: Advanced technology development for breakthroughs in fuels and chemicals production

Authors
Toone, E; Winde, HD
MLA Citation
Toone, E, and Winde, HD. "Energy biotechnology in 2013: Advanced technology development for breakthroughs in fuels and chemicals production." Current Opinion in Biotechnology 24.3 (2013): 367-368.
Source
scival
Published In
Current Opinion in Biotechnology
Volume
24
Issue
3
Publish Date
2013
Start Page
367
End Page
368
DOI
10.1016/j.copbio.2013.04.009

An enthalpic basis of additivity in biphenyl hydroxamic acid ligands for stromelysin-1.

Fragment based drug discovery remains a successful tool for pharmaceutical lead discovery. Although based upon the principle of thermodynamic additivity, the underlying thermodynamic basis is poorly understood. A thermodynamic additivity analysis was performed using stromelysin-1 and a series of biphenyl hydroxamate ligands identified through fragment additivity. Our studies suggest that, in this instance, additivity arises from enthalpic effects, while interaction entropies are unfavorable; this thermodynamic behavior is masked by proton transfer. Evaluation of the changes in constant pressure heat capacities during binding suggest that solvent exclusion from the binding site does not account for the dramatic affinity enhancements observed.

Authors
Wilfong, EM; Du, Y; Toone, EJ
MLA Citation
Wilfong, EM, Du, Y, and Toone, EJ. "An enthalpic basis of additivity in biphenyl hydroxamic acid ligands for stromelysin-1." Bioorg Med Chem Lett 22.20 (October 15, 2012): 6521-6524.
PMID
22985855
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry Letters
Volume
22
Issue
20
Publish Date
2012
Start Page
6521
End Page
6524
DOI
10.1016/j.bmcl.2012.05.032

A general and efficient cantilever functionalization technique for AFM molecular recognition studies.

Atomic force microscopy (AFM) is a versatile technique for the investigation of noncovalent molecular associations between ligand-substrate pairs. Surface modification of silicon nitride AFM cantilevers is most commonly achieved using organic trialkoxysilanes. However, susceptibility of the Si−O bond to hydrolysis and formation of polymeric aggregates diminishes attractiveness of this method for AFM studies. Attachment techniques that facilitate immobilization of a wide variety of organic and biological molecules via the stable Si−C bond on silicon nitride cantilevers would be of great value to the field of molecular recognition force spectroscopy. Here, we report (1) the formation of stable, highly oriented monolayers on the tip of silicon nitride cantilevers and (2) demonstrate their utility in the investigation of noncovalent protein-ligand interactions using molecular recognition force spectroscopy. The monolayers are formed through hydrosilylation of hydrogen-terminated silicon nitride AFM probes using a protected α-amino-ω-alkene. This approach facilitates the subsequent conjugation of biomolecules. The resulting biomolecules are bound to the tip by a strong Si−C bond, completely uniform with regard to both epitope density and substrate orientation, and highly suitable for force microscopy studies. We show that this attachment technique can be used to measure the unbinding profiles of tip-immobilized lactose and surface-immobilized galectin-3. Overall, the proposed technique is general, operationally simple, and can be expanded to anchor a wide variety of epitopes to a silicon nitride cantilever using a stable Si−C bond.

Authors
Bowers, CM; Carlson, DA; Shestopalov, AA; Clark, RL; Toone, EJ
MLA Citation
Bowers, CM, Carlson, DA, Shestopalov, AA, Clark, RL, and Toone, EJ. "A general and efficient cantilever functionalization technique for AFM molecular recognition studies." Biopolymers 97.10 (October 2012): 761-765.
PMID
22806495
Source
pubmed
Published In
Biopolymers
Volume
97
Issue
10
Publish Date
2012
Start Page
761
End Page
765
DOI
10.1002/bip.22061

Multicomponent patterning of indium tin oxide.

We report a versatile functionalization and pattering technique that permits multicomponent pattern-specific modification of indium tin oxide (ITO) with organic species. The method relies on a bilayered molecular system that simultaneously protects ITO from degradation and provides uniform chemical functionality suitable for further elaboration. Pattern-specific modification is achieved via specific reaction between functionality on an elastomeric stamp and functionality of cognate reactivity at the surface of a bilayered molecular substrate. We demonstrate that a single molecular system in a combination with different printing approaches can be used to immobilize multiple organic functionalities with exquisite spatial control on a single ITO surface. Our study provides the first general approach that permits patterning and functionalization of ITO with different molecules using a single set of printing conditions and materials.

Authors
Bowers, CM; Shestopalov, AA; Clark, RL; Toone, EJ
MLA Citation
Bowers, CM, Shestopalov, AA, Clark, RL, and Toone, EJ. "Multicomponent patterning of indium tin oxide." ACS Appl Mater Interfaces 4.8 (August 2012): 3932-3937.
PMID
22834789
Source
pubmed
Published In
ACS Applied Materials and Interfaces
Volume
4
Issue
8
Publish Date
2012
Start Page
3932
End Page
3937
DOI
10.1021/am300768f

Improving upon nature: active site remodeling produces highly efficient aldolase activity toward hydrophobic electrophilic substrates.

The substrate specificity of enzymes is frequently narrow and constrained by multiple interactions, limiting the use of natural enzymes in biocatalytic applications. Aldolases have important synthetic applications, but the usefulness of these enzymes is hampered by their narrow reactivity profile with unnatural substrates. To explore the determinants of substrate selectivity and alter the specificity of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, we employed structure-based mutagenesis coupled with library screening of mutant enzymes localized to the bacterial periplasm. We identified two active site mutations (T161S and S184L) that work additively to enhance the substrate specificity of this aldolase to include catalysis of retro-aldol cleavage of (4S)-2-keto-4-hydroxy-4-(2'-pyridyl)butyrate (S-KHPB). These mutations improve the value of k(cat)/K(M)(S-KHPB) by >450-fold, resulting in a catalytic efficiency that is comparable to that of the wild-type enzyme with the natural substrate while retaining high stereoselectivity. Moreover, the value of k(cat)(S-KHPB) for this mutant enzyme, a parameter critical for biocatalytic applications, is 3-fold higher than the maximal value achieved by the natural aldolase with any substrate. This mutant also possesses high catalytic efficiency for the retro-aldol cleavage of the natural substrate, KDPG, and a >50-fold improved activity for cleavage of 2-keto-4-hydroxy-octonoate, a nonfunctionalized hydrophobic analogue. These data suggest a substrate binding mode that illuminates the origin of facial selectivity in aldol addition reactions catalyzed by KDPG and 2-keto-3-deoxy-6-phosphogalactonate aldolases. Furthermore, targeting mutations to the active site provides a marked improvement in substrate selectivity, demonstrating that structure-guided active site mutagenesis combined with selection techniques can efficiently identify proteins with characteristics that compare favorably to those of naturally occurring enzymes.

Authors
Cheriyan, M; Toone, EJ; Fierke, CA
MLA Citation
Cheriyan, M, Toone, EJ, and Fierke, CA. "Improving upon nature: active site remodeling produces highly efficient aldolase activity toward hydrophobic electrophilic substrates." Biochemistry 51.8 (February 28, 2012): 1658-1668.
PMID
22316217
Source
pubmed
Published In
Biochemistry
Volume
51
Issue
8
Publish Date
2012
Start Page
1658
End Page
1668
DOI
10.1021/bi201899b

Modulation of Wnt/β-catenin signaling and proliferation by a ferrous iron chelator with therapeutic efficacy in genetically engineered mouse models of cancer.

Using a screen for Wnt/β-catenin inhibitors, a family of 8-hydroxyquinolone derivatives with in vivo anti-cancer properties was identified. Analysis of microarray data for the lead compound N-((8-hydroxy-7-quinolinyl) (4-methylphenyl)methyl)benzamide (HQBA) using the Connectivity Map database suggested that it is an iron chelator that mimics the hypoxic response. HQBA chelates Fe(2+) with a dissociation constant of ∼10(-19) M, with much weaker binding to Fe(3+) and other transition metals. HQBA inhibited proliferation of multiple cell lines in culture, and blocked the progression of established spontaneous cancers in two distinct genetically engineered mouse models of mammary cancer, MMTV-Wnt1 and MMTV-PyMT mice, without overt toxicity. HQBA may inhibit an iron-dependent factor that regulates cell-type-specific β-catenin-driven transcription. It inhibits cancer cell proliferation independently of its effect on β-catenin signaling, as it works equally well in MMTV-PyMT tumors and diverse β-catenin-independent cell lines. HQBA is a promising specific intracellular Fe(2+) chelator with activity against spontaneous mouse mammary cancers.

Authors
Coombs, GS; Schmitt, AA; Canning, CA; Alok, A; Low, ICC; Banerjee, N; Kaur, S; Utomo, V; Jones, CM; Pervaiz, S; Toone, EJ; Virshup, DM
MLA Citation
Coombs, GS, Schmitt, AA, Canning, CA, Alok, A, Low, ICC, Banerjee, N, Kaur, S, Utomo, V, Jones, CM, Pervaiz, S, Toone, EJ, and Virshup, DM. "Modulation of Wnt/β-catenin signaling and proliferation by a ferrous iron chelator with therapeutic efficacy in genetically engineered mouse models of cancer." Oncogene 31.2 (January 12, 2012): 213-225.
PMID
21666721
Source
pubmed
Published In
Oncogene: Including Oncogene Reviews
Volume
31
Issue
2
Publish Date
2012
Start Page
213
End Page
225
DOI
10.1038/onc.2011.228

Derivatives of plant phenolic compound affect the type III secretion system of Pseudomonas aeruginosa via a GacS-GacA two-component signal transduction system.

Antibiotic therapy is the most commonly used strategy to control pathogenic infections; however, it has contributed to the generation of antibiotic-resistant bacteria. To circumvent this emerging problem, we are searching for compounds that target bacterial virulence factors rather than their viability. Pseudomonas aeruginosa, an opportunistic human pathogen, possesses a type III secretion system (T3SS) as one of the major virulence factors by which it secretes and translocates T3 effector proteins into human host cells. The fact that this human pathogen also is able to infect several plant species led us to screen a library of phenolic compounds involved in plant defense signaling and their derivatives for novel T3 inhibitors. Promoter activity screening of exoS, which encodes a T3-secreted toxin, identified two T3 inhibitors and two T3 inducers of P. aeruginosa PAO1. These compounds alter exoS transcription by affecting the expression levels of the regulatory small RNAs RsmY and RsmZ. These two small RNAs are known to control the activity of carbon storage regulator RsmA, which is responsible for the regulation of the key T3SS regulator ExsA. As RsmY and RsmZ are the only targets directly regulated by GacA, our results suggest that these phenolic compounds affect the expression of exoS through the GacSA-RsmYZ-RsmA-ExsA regulatory pathway.

Authors
Yamazaki, A; Li, J; Zeng, Q; Khokhani, D; Hutchins, WC; Yost, AC; Biddle, E; Toone, EJ; Chen, X; Yang, C-H
MLA Citation
Yamazaki, A, Li, J, Zeng, Q, Khokhani, D, Hutchins, WC, Yost, AC, Biddle, E, Toone, EJ, Chen, X, and Yang, C-H. "Derivatives of plant phenolic compound affect the type III secretion system of Pseudomonas aeruginosa via a GacS-GacA two-component signal transduction system." Antimicrob Agents Chemother 56.1 (January 2012): 36-43.
PMID
21968370
Source
pubmed
Published In
Antimicrobial agents and chemotherapy
Volume
56
Issue
1
Publish Date
2012
Start Page
36
End Page
43
DOI
10.1128/AAC.00732-11

Soft lithographic functionalization and patterning oxide-free silicon and germanium.

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. Microcontact printing (μCP) is a soft-lithographic technique for patterning SAMs on myriad surfaces. Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 μm. In contrast to traditional printing, inkless μCP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (<200 nm) features. However, up till now, inkless μCP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation. Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many μCP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Authors
Bowers, CM; Toone, EJ; Clark, RL; Shestopalov, AA
MLA Citation
Bowers, CM, Toone, EJ, Clark, RL, and Shestopalov, AA. "Soft lithographic functionalization and patterning oxide-free silicon and germanium. (Published online)" J Vis Exp 58 (December 16, 2011).
PMID
22214997
Source
pubmed
Published In
Journal of Visualized Experiments
Issue
58
Publish Date
2011
DOI
10.3791/3478

Preface

Authors
Toone, EJ
MLA Citation
Toone, EJ. Preface. December 1, 2011.
Source
scopus
Volume
78 1
Publish Date
2011

Directed evolution of a pyruvate aldolase to recognize a long chain acyl substrate.

The use of biological catalysts for industrial scale synthetic chemistry is highly attractive, given their cost effectiveness, high specificity that obviates the need for protecting group chemistry, and the environmentally benign nature of enzymatic procedures. Here we evolve the naturally occurring 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Thermatoga maritima and Escherichia coli, into enzymes that recognize a nonfunctionalized electrophilic substrate, 2-keto-4-hydroxyoctonoate (KHO). Using an in vivo selection based on pyruvate auxotrophy, mutations were identified that lower the K(M) value up to 100-fold in E. coli KDPG aldolase, and that enhance the efficiency of retro-aldol cleavage of KHO by increasing the value of k(cat)/K(M) up to 25-fold in T. maritima KDPG aldolase. These data indicate that numerous mutations distal from the active site contribute to enhanced 'uniform binding' of the substrates, which is the first step in the evolution of novel catalytic activity.

Authors
Cheriyan, M; Walters, MJ; Kang, BD; Anzaldi, LL; Toone, EJ; Fierke, CA
MLA Citation
Cheriyan, M, Walters, MJ, Kang, BD, Anzaldi, LL, Toone, EJ, and Fierke, CA. "Directed evolution of a pyruvate aldolase to recognize a long chain acyl substrate." Bioorg Med Chem 19.21 (November 1, 2011): 6447-6453.
PMID
21944547
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry
Volume
19
Issue
21
Publish Date
2011
Start Page
6447
End Page
6453
DOI
10.1016/j.bmc.2011.08.056

A multidisciplinary approach to probing enthalpy-entropy compensation and the interfacial mobility model.

In recent years, interfacial mobility has gained popularity as a model with which to rationalize both affinity in ligand binding and the often observed phenomenon of enthalpy-entropy compensation. While protein contraction and reduced mobility, as demonstrated by computational and NMR techniques respectively, have been correlated to entropies of binding for a variety of systems, to our knowledge, Raman difference spectroscopy has never been included in these analyses. Here, nonresonance Raman difference spectroscopy, isothermal titration calorimetry, and X-ray crystallography were utilized to correlate protein contraction, as demonstrated by an increase in protein interior packing and decreased residual protein movement, with trends of enthalpy-entropy compensation. These results are in accord with the interfacial mobility model and lend additional credence to this view of protein activity.

Authors
Wilfong, EM; Kogiso, Y; Muthukrishnan, S; Kowatz, T; Du, Y; Bowie, A; Naismith, JH; Hadad, CM; Toone, EJ; Gustafson, TL
MLA Citation
Wilfong, EM, Kogiso, Y, Muthukrishnan, S, Kowatz, T, Du, Y, Bowie, A, Naismith, JH, Hadad, CM, Toone, EJ, and Gustafson, TL. "A multidisciplinary approach to probing enthalpy-entropy compensation and the interfacial mobility model." J Am Chem Soc 133.30 (August 3, 2011): 11515-11523.
PMID
21692482
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
133
Issue
30
Publish Date
2011
Start Page
11515
End Page
11523
DOI
10.1021/ja1098287

Lipooligosaccharide is required for the generation of infectious elementary bodies in Chlamydia trachomatis.

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are the main lipid components of bacterial outer membranes and are essential for cell viability in most Gram-negative bacteria. Here we show that small molecule inhibitors of LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase], the enzyme that catalyzes the first committed step in the biosynthesis of lipid A, block the synthesis of LOS in the obligate intracellular bacterial pathogen Chlamydia trachomatis. In the absence of LOS, Chlamydia remains viable and establishes a pathogenic vacuole ("inclusion") that supports robust bacterial replication. However, bacteria grown under these conditions were no longer infectious. In the presence of LpxC inhibitors, replicative reticulate bodies accumulated in enlarged inclusions but failed to express selected late-stage proteins and transition to elementary bodies, a Chlamydia developmental form that is required for invasion of mammalian cells. These findings suggest the presence of an outer membrane quality control system that regulates Chlamydia developmental transition to infectious elementary bodies and highlights the potential application of LpxC inhibitors as unique class of antichlamydial agents.

Authors
Nguyen, BD; Cunningham, D; Liang, X; Chen, X; Toone, EJ; Raetz, CRH; Zhou, P; Valdivia, RH
MLA Citation
Nguyen, BD, Cunningham, D, Liang, X, Chen, X, Toone, EJ, Raetz, CRH, Zhou, P, and Valdivia, RH. "Lipooligosaccharide is required for the generation of infectious elementary bodies in Chlamydia trachomatis." Proc Natl Acad Sci U S A 108.25 (June 21, 2011): 10284-10289.
PMID
21628561
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
108
Issue
25
Publish Date
2011
Start Page
10284
End Page
10289
DOI
10.1073/pnas.1107478108

Soft-lithographic approach to functionalization and nanopatterning oxide-free silicon.

We report a simple, reliable high-throughput method for patterning passivated silicon with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The approach completely protects silicon from chemical oxidation, provides precise control over the shape and size of the patterned features in the 100 nm domain, and gives rapid, ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules.

Authors
Shestopalov, AA; Morris, CJ; Vogen, BN; Hoertz, A; Clark, RL; Toone, EJ
MLA Citation
Shestopalov, AA, Morris, CJ, Vogen, BN, Hoertz, A, Clark, RL, and Toone, EJ. "Soft-lithographic approach to functionalization and nanopatterning oxide-free silicon." Langmuir 27.10 (May 17, 2011): 6478-6485.
PMID
21520913
Source
pubmed
Published In
Langmuir
Volume
27
Issue
10
Publish Date
2011
Start Page
6478
End Page
6485
DOI
10.1021/la200373g

Patterning NHS-terminated SAMs on germanium.

Here we report a simple, robust approach to patterning functional SAMs on germanium. The protocol relies on catalytic soft-lithographic pattern transfer from an elastomeric stamp bearing pendant immobilized sulfonic acid moieties to an NHS-functionalized bilayer molecular system comprising a primary ordered alkyl monolayer and a reactive ester secondary overlayer. The catalytic polyurethane-acrylate stamp was used to form micrometer-scale features of chemically distinct SAMs on germanium. The methodology represents the first example of patterned SAMs on germanium, a semiconductor material.

Authors
Morris, CJ; Shestopalov, AA; Gold, BH; Clark, RL; Toone, EJ
MLA Citation
Morris, CJ, Shestopalov, AA, Gold, BH, Clark, RL, and Toone, EJ. "Patterning NHS-terminated SAMs on germanium." Langmuir 27.10 (May 17, 2011): 6486-6489.
PMID
21504221
Source
pubmed
Published In
Langmuir
Volume
27
Issue
10
Publish Date
2011
Start Page
6486
End Page
6489
DOI
10.1021/la200374k

Thermodynamic characterization of the binding interaction between the histone demethylase LSD1/KDM1 and CoREST.

Flavin-dependent histone demethylases catalyze the posttranslational oxidative demethylation of mono- and dimethylated lysine residues, producing formaldehyde and hydrogen peroxide in addition to the corresponding demethylated protein. In vivo, histone demethylase LSD1 (KDM1; BCH110) is a component of the multiprotein complex that includes histone deacetylases (HDAC 1 and 2) and the scaffolding protein CoREST. Although little is known about the affinities of or the structural basis for the interaction between CoREST and HDACs, the structure of CoREST(286-482) bound to an α-helical coiled-coil tower domain within LSD1 has recently been reported. Given the significance of CoREST in directing demethylation to specific nucleosomal substrates, insight into the molecular basis of the interaction between CoREST and LSD1 may suggest a new means of inhibiting LSD1 activity by misdirecting the enzyme away from nucleosomal substrates. Toward this end, isothermal titration calorimetry studies were conducted to determine the affinity and thermodynamic parameters characterizing the binding interaction between LSD1 and CoREST(286-482). The proteins tightly interact in a 1:1 stoichiometry with a dissociation constant (K(d)) of 15.9 ± 2.07 nM, and their binding interaction is characterized by a favorable enthalpic contribution near room temperature with a smaller entropic penalty at pH 7.4. Additionally, one proton is transferred from the buffer to the heterodimeric complex at pH 7.4. From the temperature dependence of the enthalpy change of interaction, a constant-pressure heat capacity change (ΔC(p)) of the interaction was determined to be -0.80 ± 0.01 kcal mol(-1) K(-1). Notably, structure-driven truncation of CoREST revealed that the central binding determinant lies within the segment of residues 293-380, also known as the CoREST "linker" region, which is a central isolated helix that interacts with the LSD1 coiled-coil tower domain to create a triple-helical bundle. Thermodynamic parameters obtained from the binding between LSD1 and the linker region of CoREST are similar to those obtained from the interaction between LSD1 and CoREST(286-482). These results provide a framework for understanding the molecular basis of protein-protein interactions that govern nucleosomal demethylation.

Authors
Hwang, S; Schmitt, AA; Luteran, AE; Toone, EJ; McCafferty, DG
MLA Citation
Hwang, S, Schmitt, AA, Luteran, AE, Toone, EJ, and McCafferty, DG. "Thermodynamic characterization of the binding interaction between the histone demethylase LSD1/KDM1 and CoREST." Biochemistry 50.4 (February 1, 2011): 546-557.
PMID
21142040
Source
pubmed
Published In
Biochemistry
Volume
50
Issue
4
Publish Date
2011
Start Page
546
End Page
557
DOI
10.1021/bi101776t

Species-specific and inhibitor-dependent conformations of LpxC: implications for antibiotic design.

LpxC is an essential enzyme in the lipid A biosynthetic pathway in gram-negative bacteria. Several promising antimicrobial lead compounds targeting LpxC have been reported, though they typically display a large variation in potency against different gram-negative pathogens. We report that inhibitors with a diacetylene scaffold effectively overcome the resistance caused by sequence variation in the LpxC substrate-binding passage. Compound binding is captured in complex with representative LpxC orthologs, and structural analysis reveals large conformational differences that mostly reflect inherent molecular features of distinct LpxC orthologs, whereas ligand-induced structural adaptations occur at a smaller scale. These observations highlight the need for a molecular understanding of inherent structural features and conformational plasticity of LpxC enzymes for optimizing LpxC inhibitors as broad-spectrum antibiotics against gram-negative infections.

Authors
Lee, C-J; Liang, X; Chen, X; Zeng, D; Joo, SH; Chung, HS; Barb, AW; Swanson, SM; Nicholas, RA; Li, Y; Toone, EJ; Raetz, CRH; Zhou, P
MLA Citation
Lee, C-J, Liang, X, Chen, X, Zeng, D, Joo, SH, Chung, HS, Barb, AW, Swanson, SM, Nicholas, RA, Li, Y, Toone, EJ, Raetz, CRH, and Zhou, P. "Species-specific and inhibitor-dependent conformations of LpxC: implications for antibiotic design." Chem Biol 18.1 (January 28, 2011): 38-47.
PMID
21167751
Source
pubmed
Published In
Chemistry and Biology
Volume
18
Issue
1
Publish Date
2011
Start Page
38
End Page
47
DOI
10.1016/j.chembiol.2010.11.011

Syntheses, structures and antibiotic activities of LpxC inhibitors based on the diacetylene scaffold.

Compounds inhibiting LpxC in the lipid A biosynthetic pathway are promising leads for novel antibiotics against multidrug-resistant Gram-negative pathogens. We report the syntheses and structural and biochemical characterizations of LpxC inhibitors based on a diphenyl-diacetylene (1,4-diphenyl-1,3-butadiyne) threonyl-hydroxamate scaffold. These studies provide a molecular interpretation for the differential antibiotic activities of compounds with a substituted distal phenyl ring as well as the absolute stereochemical requirement at the C2, but not C3, position of the threonyl group.

Authors
Liang, X; Lee, C-J; Chen, X; Chung, HS; Zeng, D; Raetz, CRH; Li, Y; Zhou, P; Toone, EJ
MLA Citation
Liang, X, Lee, C-J, Chen, X, Chung, HS, Zeng, D, Raetz, CRH, Li, Y, Zhou, P, and Toone, EJ. "Syntheses, structures and antibiotic activities of LpxC inhibitors based on the diacetylene scaffold." Bioorg Med Chem 19.2 (January 15, 2011): 852-860.
PMID
21194954
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry
Volume
19
Issue
2
Publish Date
2011
Start Page
852
End Page
860
DOI
10.1016/j.bmc.2010.12.017

Exploring variation in binding of Protein A and Protein G to immunoglobulin type G by isothermal titration calorimetry

Bacterial Protein A (PrtA) and Protein G (PrtG) are widely used for affinity purification of antibodies. An understanding of how PrtA and PrtG bind to different isotypes of immunoglobulin type G (IgG) and to their corresponding Fc fragments is essential for the development of PrtA and PrtG mimetic ligands and for the establishment of generic processes for the purification of various antibodies. In this paper, the interactions between the two IgG-binding proteins and IgG of two different subclasses, IgG1 and IgG4, as well as their analogous Fc fragments have been studied by isothermal titration calorimetry. The results indicate that both protein ligands bind IgG and Fc fragments strongly with Ka values in the range of 10 7-10 8 M -1 and for both ligands, the interaction with both IgG isotypes is enthalpically driven though entropically unfavorable. Moreover, variation in the standard entropic and standard enthalpic contribution to binding between the two isotypes as well as between IgG and Fc fragment implies that the specific interaction with PrtA varies according to IgG isotype. In contrast to PrtA, PrtG bound to F(ab') 2 fragment with a Ka value of 5.1 × 10 5 M -1; thus underscoring the usefulness of PrtA as a preferred ligand for generic antibody purification processes. Copyright © 2011 John Wiley & Sons, Ltd. Copyright © 2011 John Wiley & Sons, Ltd.

Authors
Lund, LN; Christensen, T; Toone, E; Houen, G; Staby, A; Hilaire, PMS
MLA Citation
Lund, LN, Christensen, T, Toone, E, Houen, G, Staby, A, and Hilaire, PMS. "Exploring variation in binding of Protein A and Protein G to immunoglobulin type G by isothermal titration calorimetry." Journal of Molecular Recognition 24.6 (2011): 945-952.
PMID
22038801
Source
scival
Published In
Journal of Molecular Recognition
Volume
24
Issue
6
Publish Date
2011
Start Page
945
End Page
952
DOI
10.1002/jmr.1140

Advances in enzymology and related areas of molecular biology. Preface.

Authors
Toone, EJ
MLA Citation
Toone, EJ. "Advances in enzymology and related areas of molecular biology. Preface." Adv Enzymol Relat Areas Mol Biol 78 (2011): ix-xi.
PMID
22220470
Source
pubmed
Published In
Advances in enzymology and related areas of molecular biology
Volume
78
Publish Date
2011
Start Page
ix
End Page
xi

Bacterial infection remains a leading cause of death in both Western and developing world. Preface.

Authors
Toone, EJ
MLA Citation
Toone, EJ. "Bacterial infection remains a leading cause of death in both Western and developing world. Preface." Adv Enzymol Relat Areas Mol Biol 77 (2011): xi-xiii.
PMID
21692365
Source
pubmed
Published In
Advances in enzymology and related areas of molecular biology
Volume
77
Publish Date
2011
Start Page
xi
End Page
xiii

Preface

Authors
Broder, J
MLA Citation
Broder, J. "Preface." 2011. xi-xii.
Source
scival
Publish Date
2011
Start Page
xi
End Page
xii
DOI
10.1016/B978-1-4160-6113-7.10024-9

Identification and inhibitory properties of a novel Ca(2+)/calmodulin antagonist.

We developed a high-throughput yeast-based assay to screen for chemical inhibitors of Ca(2+)/calmodulin-dependent kinase pathways. After screening two small libraries, we identified the novel antagonist 125-C9, a substituted ethyleneamine. In vitro kinase assays confirmed that 125-C9 inhibited several calmodulin-dependent kinases (CaMKs) competitively with Ca(2+)/calmodulin (Ca(2+)/CaM). This suggested that 125-C9 acted as an antagonist for Ca(2+)/CaM rather than for CaMKs. We confirmed this hypothesis by showing that 125-C9 binds directly to Ca(2+)/CaM using isothermal titration calorimetry. We further characterized binding of 125-C9 to Ca(2+)/CaM and compared its properties with those of two well-studied CaM antagonists: trifluoperazine (TFP) and W-13. Isothermal titration calorimetry revealed that binding of 125-C9 to CaM is absolutely Ca(2+)-dependent, likely occurs with a stoichiometry of five 125-C9 molecules to one CaM molecule, and involves an exchange of two protons at pH 7.0. Binding of 125-C9 is driven overall by entropy and appears to be competitive with TFP and W-13, which is consistent with occupation of similar binding sites. To test the effects of 125-C9 in living cells, we evaluated mitogen-stimulated re-entry of quiescent cells into proliferation and found similar, although slightly better, levels of inhibition by 125-C9 than by TFP and W-13. Our results not only define a novel Ca(2+)/CaM inhibitor but also reveal that chemically unique CaM antagonists can bind CaM by distinct mechanisms but similarly inhibit cellular actions of CaM.

Authors
Colomer, J; Schmitt, AA; Toone, EJ; Means, AR
MLA Citation
Colomer, J, Schmitt, AA, Toone, EJ, and Means, AR. "Identification and inhibitory properties of a novel Ca(2+)/calmodulin antagonist." Biochemistry 49.19 (May 18, 2010): 4244-4254.
Website
http://hdl.handle.net/10161/4003
PMID
20392081
Source
pubmed
Published In
Biochemistry
Volume
49
Issue
19
Publish Date
2010
Start Page
4244
End Page
4254
DOI
10.1021/bi1001213

Catalytic microcontact printing on chemically functionalized H-terminated silicon.

We report a novel inkless soft lithographic fabrication protocol that permits uniform parallel patterning of hydrogen-terminated silicon surfaces using catalytic elastomeric stamps. Pattern transfer is achieved catalytically via reaction between sulfonic acid moieties covalently bound to an elastomeric stamp and a Boc-functionalized SAM grafted to passivated silicon. The approach represents the first example of a soft lithographic printing technique that creates patterns of chemically distinctive SAMs on oxide-free silicon substrates.

Authors
Shestopalov, AA; Clark, RL; Toone, EJ
MLA Citation
Shestopalov, AA, Clark, RL, and Toone, EJ. "Catalytic microcontact printing on chemically functionalized H-terminated silicon." Langmuir 26.3 (February 2, 2010): 1449-1451.
Website
http://hdl.handle.net/10161/4089
PMID
19950970
Source
pubmed
Published In
Langmuir
Volume
26
Issue
3
Publish Date
2010
Start Page
1449
End Page
1451
DOI
10.1021/la903465f

A single step purification for autolytic zinc proteinases.

We describe a novel single-step method for the purification of stromelysin-1 catalytic domain (SCD) via immobilized metal affinity chromatography under denaturing conditions that inhibit proteolytic activity followed by on-column refolding and spontaneous autolysis of the fusion peptide to yield pure, active stromelysin-1 catalytic domain. The methodology provides a general approach for the rapid purification of large quantities of zinc proteinases.

Authors
Wilfong, EM; Locklear, U; Toone, EJ
MLA Citation
Wilfong, EM, Locklear, U, and Toone, EJ. "A single step purification for autolytic zinc proteinases." Bioorg Med Chem Lett 20.1 (January 1, 2010): 280-282.
PMID
19942433
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry Letters
Volume
20
Issue
1
Publish Date
2010
Start Page
280
End Page
282
DOI
10.1016/j.bmcl.2009.10.114

Inkless microcontact printing on SAMs of Boc- and TBS-protected thiols.

We report a new inkless catalytic muCP technique that achieves accurate, fast, and complete pattern reproduction on SAMs of Boc- and TBS-protected thiols immobilized on gold using a polyurethane-acrylate stamp functionalized with covalently bound sulfonic acids. Pattern transfer is complete at room temperature just after one minute of contact and renders sub-200 nm size structures of chemically differentiated SAMs.

Authors
Shestopalov, AA; Clark, RL; Toone, EJ
MLA Citation
Shestopalov, AA, Clark, RL, and Toone, EJ. "Inkless microcontact printing on SAMs of Boc- and TBS-protected thiols." Nano Lett 10.1 (January 2010): 43-46.
Website
http://hdl.handle.net/10161/4097
PMID
19950928
Source
pubmed
Published In
Nano Letters
Volume
10
Issue
1
Publish Date
2010
Start Page
43
End Page
46
DOI
10.1021/nl902588d

Preface

Authors
Toone, EJ
MLA Citation
Toone, EJ. "Preface." Advances in Enzymology and Related Areas of Molecular Biology 77 1 (2010): xi-xiii.
Source
scival
Published In
Advances in enzymology and related areas of molecular biology
Volume
77 1
Publish Date
2010
Start Page
xi
End Page
xiii

Minimizing tip-sample contact force in automated atomic force microscope based force spectroscopy

In atomic force microscope based force spectroscopy, it is often necessary to minimize the tip-sample contact force. While it is possible to control the contact force using force feedback, this method is susceptible to sensor drift and is often underutilized due to the noise associated with the feedback process. Here we present a method to control the tip-sample contact force for repeated pulling cycles without relying on force feedback or tedious user-controlled z-stage step increments. The custom pulling program uses the data recorded during the previous retraction cycle to automatically reposition the sample surface to account for changes in topography and system drift. Using this method we were able to complete 250 automated pulling cycles, 76% of which had evidence of tip-sample contact. Of those pulling cycles with tip-sample contact, the average contact force was 83 pN, with the maximum contact force not exceeding 292 pN. Copyright © 2009 by ASME.

Authors
Rivera, M; Morris, C; Carlson, D; Toone, EJ; Cole, DG; Clark, RL
MLA Citation
Rivera, M, Morris, C, Carlson, D, Toone, EJ, Cole, DG, and Clark, RL. "Minimizing tip-sample contact force in automated atomic force microscope based force spectroscopy." Proceedings of the ASME International Design Engineering Technical Conferences and Computers and Information in Engineering Conference 2009, DETC2009 6 (2010): 731-736.
Source
scival
Published In
Proceedings of the ASME International Design Engineering Technical Conferences and Computers and Information in Engineering Conference 2009, DETC2009
Volume
6
Publish Date
2010
Start Page
731
End Page
736
DOI
10.1115/DETC2009-87378

Forcing a closer look

Authors
Toone, EJ
MLA Citation
Toone, EJ. "Forcing a closer look." Biopolymers 93.1 (2010): iii-.
Source
scival
Published In
Biopolymers
Volume
93
Issue
1
Publish Date
2010
Start Page
iii
DOI
10.1002/bip.21330

In situ growth of a stoichiometric PEG-like conjugate at a protein's N-terminus with significantly improved pharmacokinetics.

The challenge in the synthesis of protein-polymer conjugates for biological applications is to synthesize a stoichiometric (typically 1:1) conjugate of the protein with a monodisperse polymer, with good retention of protein activity, significantly improved pharmacokinetics and increased bioavailability, and hence improved in vivo efficacy. Here we demonstrate, using myoglobin as an example, a general route to grow a PEG-like polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) [poly(OEGMA)], with low polydispersity and high yield, solely from the N-terminus of the protein by in situ atom transfer radical polymerization (ATRP) under aqueous conditions, to yield a site-specific (N-terminal) and stoichiometric conjugate (1:1). Notably, the myoglobin-poly(OEGMA) conjugate [hydrodynamic radius (R(h)): 13 nm] showed a 41-fold increase in its blood exposure compared to the protein (R(h): 1.7 nm) after IV administration to mice, thereby demonstrating that comb polymers that present short oligo(ethylene glycol) side chains are a class of PEG-like polymers that can significantly improve the pharmacological properties of proteins. We believe that this approach to the synthesis of N-terminal protein conjugates of poly(OEGMA) may be applicable to a large subset of protein and peptide drugs, and thereby provide a general methodology for improvement of their pharmacological profiles.

Authors
Gao, W; Liu, W; Mackay, JA; Zalutsky, MR; Toone, EJ; Chilkoti, A
MLA Citation
Gao, W, Liu, W, Mackay, JA, Zalutsky, MR, Toone, EJ, and Chilkoti, A. "In situ growth of a stoichiometric PEG-like conjugate at a protein's N-terminus with significantly improved pharmacokinetics." Proc Natl Acad Sci U S A 106.36 (September 8, 2009): 15231-15236.
PMID
19706892
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
36
Publish Date
2009
Start Page
15231
End Page
15236
DOI
10.1073/pnas.0904378106

Eyedrops containing SA9000 prodrugs result in sustained reductions in intraocular pressure in rabbits.

AIM: Poor topical bioavailability and ocular irritation have impeded the development of the diuretic, ethacrynic acid (ECA) as a clinically useful ocular hypotensive for the treatment of glaucoma. Thus, the development of analogs and prodrugs of analogs with improved ocular penetration, potency, and tolerability is required. The aim of this work is to evaluate the corneal penetration and ocular distribution of SA9000, an ECA analog. Novel SA9000 prodrugs intended to further improve ocular pharmacodynamic effect were also evaluated. RESULTS: SA9000 penetrated porcine corneas more effectively than ECA in corneal diffusion studies. In vivo studies in Dutch-belted (DB) rabbits indicated that topical application of a single dose (0.3%) of SA9000 could significantly reduce intraocular pressure (IOP) (approximately 25% vs. fellow untreated eye) but caused significant conjunctival hyperemia. Since this hyperemia was likely the result of its inherent thiol reactivity, SA9000 was formulated with equimolar cysteine, an exogenous thiol donor. The administration of increasing SA9000-cysteine adduct concentrations (0.3%, 0.6%, 0.9%) demonstrated that they cause less ocular irritation than unadducted SA9000 but could still significantly reduce IOP (0.3%: 8.7 +/- 2%; 0.6%: 14.4 +/- 5%; 0.9%: 23.3 +/- 4.4%) versus untreated contralateral control eyes. CONCLUSIONS: These data suggest that novel thiol donor adduction can improve the ocular bioavailability and tolerability of SA9000. SA9000-cysteine prodrugs may represent a new option for the topical treatment of glaucoma.

Authors
Arnold, JJ; Choksi, Y; Chen, X; Shimazaki, A; Hatten, J; Toone, EJ; Epstein, DL; Challa, P
MLA Citation
Arnold, JJ, Choksi, Y, Chen, X, Shimazaki, A, Hatten, J, Toone, EJ, Epstein, DL, and Challa, P. "Eyedrops containing SA9000 prodrugs result in sustained reductions in intraocular pressure in rabbits." J Ocul Pharmacol Ther 25.3 (June 2009): 179-186.
PMID
19456251
Source
pubmed
Published In
Journal of Ocular Pharmacology and Therapeutics
Volume
25
Issue
3
Publish Date
2009
Start Page
179
End Page
186
DOI
10.1089/jop.2008.0107

C-nitroso donors of nitric oxide.

A complete understanding of the biological activity of nitric oxide (NO) is complicated by the different reactivity profiles of its various species and by the often complex decomposition behavior of the NO progenitors in common use. Here, we report that appropriately substituted C-nitroso compounds act solely as donors of neutral nitric oxide through a first-order homolytic C-N bond scission to release up to 88% nitric oxide in DMSO at 25 degrees C. The reaction produces a carbon radical, and the yield of nitric oxide is dependent on the availability of radical traps. C-Nitroso compounds are sources of biologically active neutral NO and display potent NO bioactivity in a rabbit aortic ring assay.

Authors
Chakrapani, H; Bartberger, MD; Toone, EJ
MLA Citation
Chakrapani, H, Bartberger, MD, and Toone, EJ. "C-nitroso donors of nitric oxide." J Org Chem 74.4 (February 20, 2009): 1450-1453.
PMID
19146387
Source
pubmed
Published In
The Journal of Organic Chemistry
Volume
74
Issue
4
Publish Date
2009
Start Page
1450
End Page
1453
DOI
10.1021/jo802517t

Natural product glycosyltransferases: Properties and applications

Authors
Gavin, JW; Jon, ST; Toone, EJ
MLA Citation
Gavin, JW, Jon, ST, and Toone, EJ. "Natural product glycosyltransferases: Properties and applications." Advances in Enzymology and Related Areas of Molecular Biology 76 (2009): 55-119.
PMID
18990828
Source
scival
Published In
Advances in enzymology and related areas of molecular biology
Volume
76
Publish Date
2009
Start Page
55
End Page
119

Minimizing tip-sample contact force in automated atomic force microscope based force spectroscopy

In atomic force microscope based force spectroscopy, it is often necessary to minimize the tip-sample contact force. While it is possible to control the contact force using force feedback, this method is susceptible to sensor drift and is often underutilized due to the noise associated with the feedback process. Here we present a method to control the tip-sample contact force for repeated pulling cycles without relying on force feedback or tedious user-controlled z-stage step increments. The custom pulling program uses the data recorded during the previous retraction cycle to automatically reposition the sample surface to account for changes in topography and system drift. Using this method we were able to complete 250 automated pulling cycles, 76% of which had evidence of tip-sample contact. Of those pulling cycles with tip-sample contact, the average contact force was 83 pN, with the maximum contact force not exceeding 292 pN. Copyright © 2009 by ASME.

Authors
Rivera, M; Morris, C; Carlson, D; Toone, EJ; Cole, DG; Clark, RL
MLA Citation
Rivera, M, Morris, C, Carlson, D, Toone, EJ, Cole, DG, and Clark, RL. "Minimizing tip-sample contact force in automated atomic force microscope based force spectroscopy." Proceedings of the ASME Design Engineering Technical Conference 6 (2009): 731-736.
Source
scival
Published In
Proceedings of the ASME Design Engineering Technical Conference
Volume
6
Publish Date
2009
Start Page
731
End Page
736
DOI
10.1115/DETC2009-87378

Advances in enzymology and related areas of molecular biology. Preface.

Authors
Toone, EJ
MLA Citation
Toone, EJ. "Advances in enzymology and related areas of molecular biology. Preface." Adv Enzymol Relat Areas Mol Biol 76 (2009): ix-x.
PMID
18990826
Source
pubmed
Published In
Advances in enzymology and related areas of molecular biology
Volume
76
Publish Date
2009
Start Page
ix
End Page
x

Identification of chemical inhibitors to human tissue transglutaminase by screening existing drug libraries.

Human tissue transglutaminase (TGM2) is a calcium-dependent crosslinking enzyme involved in the posttranslational modification of intra- and extracellular proteins and implicated in several neurodegenerative diseases. To find specific inhibitors to TGM2, two structurally diverse chemical libraries (LOPAC and Prestwick) were screened. We found that ZM39923, a Janus kinase inhibitor, and its metabolite ZM449829 were the most potent inhibitors with IC(50) of 10 and 5 nM, respectively. In addition, two other inhibitors, including tyrphostin 47 and vitamin K(3), were found to have an IC(50) in the micromolar range. These agents used in part a thiol-dependent mechanism to inhibit TGM2, consistent with the activation of TGM2 by reduction of an intramolecular disulfide bond. These inhibitors were tested in a polyglutamine-expressing Drosophila model of neurodegeneration and found to improve survival. The TGM2 inhibitors we discovered may serve as valuable lead compounds for the development of orally active TGM2 inhibitors to treat human diseases.

Authors
Lai, T-S; Liu, Y; Tucker, T; Daniel, KR; Sane, DC; Toone, E; Burke, JR; Strittmatter, WJ; Greenberg, CS
MLA Citation
Lai, T-S, Liu, Y, Tucker, T, Daniel, KR, Sane, DC, Toone, E, Burke, JR, Strittmatter, WJ, and Greenberg, CS. "Identification of chemical inhibitors to human tissue transglutaminase by screening existing drug libraries." Chem Biol 15.9 (September 22, 2008): 969-978.
PMID
18804034
Source
pubmed
Published In
Chemistry & Biology
Volume
15
Issue
9
Publish Date
2008
Start Page
969
End Page
978
DOI
10.1016/j.chembiol.2008.07.015

Characterization and crystal structure of Escherichia coli KDPGal aldolase.

2-Keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases catalyze an identical reaction differing in substrate specificity in only the configuration of a single stereocenter. However, the proteins show little sequence homology at the amino acid level. Here we investigate the determinants of substrate selectivity of these enzymes. The Escherichia coli KDPGal aldolase gene, cloned into a T7 expression vector and overexpressed in E. coli, catalyzes retro-aldol cleavage of the natural substrate, KDPGal, with values of k(cat)/K(M) and k(cat) of 1.9x10(4)M(-1)s(-1) and 4s(-1), respectively. In the synthetic direction, KDPGal aldolase efficiently catalyzes an aldol addition using a limited number of aldehyde substrates, including d-glyceraldehyde-3-phosphate (natural substrate), d-glyceraldehyde, glycolaldehyde, and 2-pyridinecarboxaldehyde. A preparative scale reaction between 2-pyridinecarboxaldehyde and pyruvate catalyzed by KDPGal aldolase produced the aldol adduct of the R stereochemistry in >99.7% ee, a result complementary to that observed using the related KDPG aldolase. The native crystal structure has been solved to a resolution of 2.4A and displays the same (alpha/beta)(8) topology, as KDPG aldolase. We have also determined a 2.1A structure of a Schiff base complex between the enzyme and its substrate. This model predicts that a single amino acid change, T161 in KDPG aldolase to V154 in KDPGal aldolase, plays an important role in determining the stereochemical course of enzyme catalysis and this prediction was borne out by site-directed mutagenesis studies. However, additional changes in the enzyme sequence are required to prepare an enzyme with both high catalytic efficiency and altered stereochemistry.

Authors
Walters, MJ; Srikannathasan, V; McEwan, AR; Naismith, JH; Fierke, CA; Toone, EJ
MLA Citation
Walters, MJ, Srikannathasan, V, McEwan, AR, Naismith, JH, Fierke, CA, and Toone, EJ. "Characterization and crystal structure of Escherichia coli KDPGal aldolase." Bioorg Med Chem 16.2 (January 15, 2008): 710-720.
PMID
17981470
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry
Volume
16
Issue
2
Publish Date
2008
Start Page
710
End Page
720
DOI
10.1016/j.bmc.2007.10.043

Inkless microcontact printing on self-assembled monolayers of fmoc-protected aminothiols.

Authors
Shestopalov, AA; Clark, RL; Toone, EJ
MLA Citation
Shestopalov, AA, Clark, RL, and Toone, EJ. "Inkless microcontact printing on self-assembled monolayers of fmoc-protected aminothiols." J Am Chem Soc 129.45 (November 14, 2007): 13818-13819.
PMID
17949005
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
129
Issue
45
Publish Date
2007
Start Page
13818
End Page
13819
DOI
10.1021/ja076226k

Mutagenesis of the phosphate-binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates.

Narrow substrate specificities often limit the use of enzymes in biocatalysis. To further the development of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase as a biocatalyst, the molecular determinants of substrate specificity were probed by mutagenesis. Our data demonstrate that S184 is located in the substrate-binding pocket and interacts with the phosphate moiety of KDPG, providing biochemical support for the binding model proposed on the basis of crystallographic data. An analysis of the substrate selectivity of the mutant enzymes indicates that alterations to the phosphate-binding site of KDPG aldolase changes the substrate selectivity. We report mutations that enhance catalysis of aldol cleavage of substrates lacking a phosphate moiety and demonstrate that electrophile reactivity correlates with the hydrophobicity of the substituted side chain. These mutations improve the selectivity for unnatural substrates as compared to KDPG by up to 2000-fold. Furthermore, the S184L KDPG aldolase mutant improves the catalytic efficiency for the synthesis of a precursor for nikkomycin by 40-fold, making it a useful biocatalyst for the preparation of fine chemicals.

Authors
Cheriyan, M; Toone, EJ; Fierke, CA
MLA Citation
Cheriyan, M, Toone, EJ, and Fierke, CA. "Mutagenesis of the phosphate-binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates." Protein Sci 16.11 (November 2007): 2368-2377.
PMID
17962400
Source
pubmed
Published In
Protein Science
Volume
16
Issue
11
Publish Date
2007
Start Page
2368
End Page
2377
DOI
10.1110/ps.073042907

Biocatalytic microcontact printing.

Immobilized biocatalytic lithography is presented as an application of soft lithography. In traditional microcontact printing, diffusion limits resolution of pattern transfer. By using an immobilized catalyst, the lateral resolution of microcontact printing would depend only on the length and flexibility of the tether (<2 nm) as opposed to diffusion (>100 nm). In the work, exonuclease reversibly immobilized on a relief-patterned stamp is used to ablate ssDNA monolayers Percent of ablation was determined via confocal fluorescence microscopy to be approximately 70%.

Authors
Snyder, PW; Johannes, MS; Vogen, BN; Clark, RL; Toone, EJ
MLA Citation
Snyder, PW, Johannes, MS, Vogen, BN, Clark, RL, and Toone, EJ. "Biocatalytic microcontact printing." J Org Chem 72.19 (September 14, 2007): 7459-7461.
PMID
17705546
Source
pubmed
Published In
The Journal of Organic Chemistry
Volume
72
Issue
19
Publish Date
2007
Start Page
7459
End Page
7461
DOI
10.1021/jo0711541

A stochastic, cantilever approach to the evaluation of solution phase thermodynamic quantities.

A cantilever device based on competitive binding of an immobilized receptor to immobilized and soluble ligand and capable of measuring solution-phase thermodynamic quantities is described. Through multiple binary queries, the device stochastically measures the probability of the formation of a bound complex between immobilized protein and immobilized ligand as a function of soluble ligand concentration. The resulting binding isotherm is described by a binding polynomial consisting of the activities of soluble and immobilized ligand and binding constants for the association of immobilized protein with free and immobilized ligand. Evaluation of the polynomial reveals an association constant for the formation of a complex between immobilized ligand and immobilized protein close to that for the formation of complex between soluble protein and soluble ligand. The methodology lays the foundation for construction of practical portable sensing devices.

Authors
Snyder, PW; Lee, G; Marszalek, PE; Clark, RL; Toone, EJ
MLA Citation
Snyder, PW, Lee, G, Marszalek, PE, Clark, RL, and Toone, EJ. "A stochastic, cantilever approach to the evaluation of solution phase thermodynamic quantities." Proc Natl Acad Sci U S A 104.8 (February 20, 2007): 2579-2584.
PMID
17307881
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
8
Publish Date
2007
Start Page
2579
End Page
2584
DOI
10.1073/pnas.0606604104

Substrate specificity of the electrophilic binding site of KDPG aldolase is altered by mutagenesis

Authors
Cheryian, M; Toone, EJ; Fierke, CA
MLA Citation
Cheryian, M, Toone, EJ, and Fierke, CA. "Substrate specificity of the electrophilic binding site of KDPG aldolase is altered by mutagenesis (Submitted)." Bioorg. Med. Chem. (2007). (Academic Article)
Source
manual
Published In
Bioorg. Med. Chem.
Publish Date
2007

Pyruvate aldolases in chiral carbon-carbon bond formation.

A procedure for the preparation of optically pure alpha-keto-gamma-hydroxy carboxylic acids through stereospecific aldol addition catalyzed by pyruvate aldolases from the Entner-Doudoroff and the DeLey-Doudoroff glycolytic pathways is described. This highly versatile fragment serves as a precursor for a variety of commonly encountered functionalities, including beta-hydroxy aldehydes and carboxylic acids, alpha-amino-gamma-hydroxy carboxylic acids and alpha,gamma-dihydroxy carboxylic acids. The protocol described here uses recombinant His6-tagged KDPG aldolase for the synthesis of (S)-4-hydroxy-2-keto-4-(2'-pyridyl)butyrate. A protocol for evaluating enantiomeric excess through formation of the gamma-lactone of the dithioacetal followed by chiral-phase gas-liquid chromatography is also described. Enzyme expression and enzymatic synthesis can be accomplished in approximately 1 week. The enzymatic aldol addition proceeds in nearly quantitative yields with enantiomeric excesses greater than 99.7%.

Authors
Walters, MJ; Toone, EJ
MLA Citation
Walters, MJ, and Toone, EJ. "Pyruvate aldolases in chiral carbon-carbon bond formation." Nat Protoc 2.7 (2007): 1825-1830.
PMID
17641651
Source
pubmed
Published In
Nature Protocols
Volume
2
Issue
7
Publish Date
2007
Start Page
1825
End Page
1830
DOI
10.1038/nprot.2007.260

Practical synthesis of fully protected globotriaose and its glycopolymers

Convenient and useful construction of a trisaccharide moiety of globotriaosyl ceramide was performed by means of modified Ogawa's protocol. In order to evaluate an efficiency of new class of glycopolymers, further chemical transformations of the trisaccharide were accomplished to afford a globotriaosyl carbohydrate monomer, and homopolymerization of the monomer by a general radical polymerization protocol gave a high-density glycopolymer in 84.3% yield after usual work-up procedures. In addition to the homopolymer, a copolymer composed of carbohydrate units and acrylamide units was also synthesized by the radical polymerization in 96.7% yield. © 2006 Elsevier Ltd. All rights reserved.

Authors
Matsuoka, K; Goshu, Y; Takezawa, Y; Mori, T; Sakamoto, J-I; Yamada, A; Onaga, T; Koyama, T; Hatano, K; Snyder, PW; Toone, EJ; Terunuma, D
MLA Citation
Matsuoka, K, Goshu, Y, Takezawa, Y, Mori, T, Sakamoto, J-I, Yamada, A, Onaga, T, Koyama, T, Hatano, K, Snyder, PW, Toone, EJ, and Terunuma, D. "Practical synthesis of fully protected globotriaose and its glycopolymers." Carbohydrate Polymers 69.2 (2007): 326-335.
Source
scival
Published In
Carbohydrate Polymers
Volume
69
Issue
2
Publish Date
2007
Start Page
326
End Page
335
DOI
10.1016/j.carbpol.2006.10.011

Preface

Authors
Toone, EJ
MLA Citation
Toone, EJ. "Preface." Advances in Enzymology and Related Areas of Molecular Biology 75 (2007): ix-x.
Source
scival
Published In
Advances in enzymology and related areas of molecular biology
Volume
75
Publish Date
2007
Start Page
ix
End Page
x

Preface

Authors
Sidebotham, D; McKee, A; Gillham, M; Levy, JH
MLA Citation
Sidebotham, D, McKee, A, Gillham, M, and Levy, JH. "Preface." Cardiothoracic Critical Care (2007): ix-ix.
Source
scival
Published In
Cardiothoracic Critical Care
Publish Date
2007
Start Page
ix
End Page
ix
DOI
10.1016/B978-075067572-7.50002-3

Binding of warfarin influences the acid-base equilibrium of H242 in sudlow site I of human serum albumin.

Sudlow Site I of human serum albumin (HSA) is located in subdomain IIA of the protein and serves as a binding cavity for a variety of ligands. In this study, the binding of warfarin (W) is examined using computational techniques and isothermal titration calorimetry (ITC). The structure of the docked warfarin anion (W-) to Site I is similar to that revealed by X-ray crystallography, with a calculated binding constant of 5.8 x 10(5) M(-1). ITC experiments (pH 7.13 and I = 0.1) carried out in three different buffers (MOPs, phosphate and Tris) reveal binding of W- is accompanied by uptake of 0.30+/-0.02 protons from the solvent. This measurement suggests that the binding of W- is stabilized by an ion-pair interaction between protonated H242 and the phenoxide group of W-.

Authors
Perry, JL; Goldsmith, MR; Williams, TR; Radack, KP; Christensen, T; Gorham, J; Pasquinelli, MA; Toone, EJ; Beratan, DN; Simon, JD
MLA Citation
Perry, JL, Goldsmith, MR, Williams, TR, Radack, KP, Christensen, T, Gorham, J, Pasquinelli, MA, Toone, EJ, Beratan, DN, and Simon, JD. "Binding of warfarin influences the acid-base equilibrium of H242 in sudlow site I of human serum albumin." Photochem Photobiol 82.5 (September 2006): 1365-1369.
PMID
16563025
Source
pubmed
Published In
Photochemistry & Photobiology
Volume
82
Issue
5
Publish Date
2006
Start Page
1365
End Page
1369
DOI
10.1562/2006-02-23-RA-811

Mechanism of the Class I KDPG aldolase.

In vivo, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible, stereospecific retro-aldol cleavage of KDPG to pyruvate and D-glyceraldehyde-3-phosphate. The enzyme is a lysine-dependent (Class I) aldolase that functions through the intermediacy of a Schiff base. Here, we propose a mechanism for this enzyme based on crystallographic studies of wild-type and mutant aldolases. The three dimensional structure of KDPG aldolase from the thermophile Thermotoga maritima was determined to 1.9A. The structure is the standard alpha/beta barrel observed for all Class I aldolases. At the active site Lys we observe clear density for a pyruvate Schiff base. Density for a sulfate ion bound in a conserved cluster of residues close to the Schiff base is also observed. We have also determined the structure of a mutant of Escherichia coli KDPG aldolase in which the proposed general acid/base catalyst has been removed (E45N). One subunit of the trimer contains density suggesting a trapped pyruvate carbinolamine intermediate. All three subunits contain a phosphate ion bound in a location effectively identical to that of the sulfate ion bound in the T. maritima enzyme. The sulfate and phosphate ions experimentally locate the putative phosphate binding site of the aldolase and, together with the position of the bound pyruvate, facilitate construction of a model for the full-length KDPG substrate complex. The model requires only minimal positional adjustments of the experimentally determined covalent intermediate and bound anion to accommodate full-length substrate. The model identifies the key catalytic residues of the protein and suggests important roles for two observable water molecules. The first water molecule remains bound to the enzyme during the entire catalytic cycle, shuttling protons between the catalytic glutamate and the substrate. The second water molecule arises from dehydration of the carbinolamine and serves as the nucleophilic water during hydrolysis of the enzyme-product Schiff base. The second water molecule may also mediate the base-catalyzed enolization required to form the carbon nucleophile, again bridging to the catalytic glutamate. Many aspects of this mechanism are observed in other Class I aldolases and suggest a mechanistically and, perhaps, evolutionarily related family of aldolases distinct from the N-acetylneuraminate lyase (NAL) family.

Authors
Fullerton, SWB; Griffiths, JS; Merkel, AB; Cheriyan, M; Wymer, NJ; Hutchins, MJ; Fierke, CA; Toone, EJ; Naismith, JH
MLA Citation
Fullerton, SWB, Griffiths, JS, Merkel, AB, Cheriyan, M, Wymer, NJ, Hutchins, MJ, Fierke, CA, Toone, EJ, and Naismith, JH. "Mechanism of the Class I KDPG aldolase." Bioorg Med Chem 14.9 (May 1, 2006): 3002-3010.
PMID
16403639
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry
Volume
14
Issue
9
Publish Date
2006
Start Page
3002
End Page
3010
DOI
10.1016/j.bmc.2005.12.022

Nitrosonium-catalyzed decomposition of s-nitrosothiols in solution: a theoretical and experimental study.

The decomposition of S-nitrosothiols (RSNO) in solution under oxidative conditions is significantly faster than can be accounted for by homolysis of the S-N bond. Here we propose a cationic chain mechanism in which nitrosation of nitrosothiol produces a nitrosated cation that, in turn, reacts with a second nitrosothiol to produce nitrosated disulfide and the NO dimer. The nitrosated disulfide acts as a source of nitrosonium for nitrosothiol nitrosation, completing the catalytic cycle. The mechanism accounts for several unexplained facets of nitrosothiol chemistry in solution, including the observation that the decomposition of an RSNO is accelerated by O(2), mixtures of O(2) and NO, and other oxidants, that decomposition is inhibited by thiols and other antioxidants, that decomposition is dependent on sulfur substitution, and that decomposition often shows nonintegral kinetic orders.

Authors
Zhao, Y-L; McCarren, PR; Houk, KN; Choi, BY; Toone, EJ
MLA Citation
Zhao, Y-L, McCarren, PR, Houk, KN, Choi, BY, and Toone, EJ. "Nitrosonium-catalyzed decomposition of s-nitrosothiols in solution: a theoretical and experimental study." J Am Chem Soc 127.31 (August 10, 2005): 10917-10924.
PMID
16076198
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
127
Issue
31
Publish Date
2005
Start Page
10917
End Page
10924
DOI
10.1021/ja050018f

A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells.

Many key regulatory proteins, including members of the Ras family of GTPases, are modified at their C terminus by a process termed prenylation. This processing is initiated by the addition of an isoprenoid lipid, and the proteins are further modified by a proteolytic event and methylation of the C-terminal prenylcysteine. Although the biological consequences of prenylation have been characterized extensively, the contributions of prenylcysteine methylation to the functions of the modified proteins are not well understood. This reaction is catalyzed by the enzyme isoprenylcysteine carboxyl methyltransferase (Icmt). Recent genetic disruption studies have provided strong evidence that blocking Icmt activity has profound consequences on oncogenic transformation. Here, we report the identification of a selective small-molecule inhibitor of Icmt, 2-[5-(3-methylphenyl)-1-octyl-1H-indol-3-yl]acetamide (cysmethynil). Cysmethynil treatment results in inhibition of cell growth in an Icmt-dependent fashion, demonstrating mechanism-based activity of the compound. Treatment of cancer cells with cysmethynil results in mislocalization of Ras and impaired epidermal growth factor signaling. In a human colon cancer cell line, cysmethynil treatment blocks anchorage-independent growth, and this effect is reversed by overexpression of Icmt. These findings provide a compelling rationale for development of Icmt inhibitors as another approach to anticancer drug development.

Authors
Winter-Vann, AM; Baron, RA; Wong, W; dela Cruz, J; York, JD; Gooden, DM; Bergo, MO; Young, SG; Toone, EJ; Casey, PJ
MLA Citation
Winter-Vann, AM, Baron, RA, Wong, W, dela Cruz, J, York, JD, Gooden, DM, Bergo, MO, Young, SG, Toone, EJ, and Casey, PJ. "A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells." Proc Natl Acad Sci U S A 102.12 (March 22, 2005): 4336-4341.
PMID
15784746
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
102
Issue
12
Publish Date
2005
Start Page
4336
End Page
4341
DOI
10.1073/pnas.0408107102

Synthesis of carbosilane dendrimers having peripheral mannose and mannobiose

The mannose monosaccharide derivative, acetylthiopropyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranoside (Man), and the mannobiose derivative, acetylthiopropyl 2,4,6-tri-O-acetyl-3-O-(2′,3′,4′, 6′-tetra-O-acetyl-α-d-mannopyranosyl)-α-d-mannopyranoside (α-1,3-Man), were synthesized respectively. These mannose derivatives were introduced into carbosilane dendrimer scaffolds of the zero and first generations. As a result, six carbosilane dendrimers were functionalized by Man and α-1,3-Man. Isothermal titration microcalorimetry was done to determine binding assay between mannose moieties of carbosilane dendrimer and concanavalin A. It was found that carbosilane dendrimers bound more efficiently to concanavalin A than free mannose (Me-α-Man) and mannobiose (Me-α-1,3-Man). © 2005 Elsevier Ltd. All rights reserved.

Authors
Mori, T; Hatano, K; Matsuoka, K; Esumi, Y; Toone, EJ; Terunuma, D
MLA Citation
Mori, T, Hatano, K, Matsuoka, K, Esumi, Y, Toone, EJ, and Terunuma, D. "Synthesis of carbosilane dendrimers having peripheral mannose and mannobiose." Tetrahedron 61.11 (2005): 2751-2760.
Source
scival
Published In
Tetrahedron
Volume
61
Issue
11
Publish Date
2005
Start Page
2751
End Page
2760
DOI
10.1016/j.tet.2005.01.090

C-nitroso compounds: synthesis, physicochemical properties and biological activities.

Because of the chemical and physical properties of nitric oxide, its effective use and delivery for therapeutic application represents a significant challenge. Accordingly, current understanding of nitric oxide biology largely stems from the use of nitric oxide prodrugs and adducts whose biological activities are based on their ability to release nitric oxide or a redox-related species. Among the structurally diverse ensemble of nitric oxide donor compounds reported to date are the C-nitroso compounds. These compounds have only recently been investigated with respect to their potential as nitric oxide donors, although they have been known and studied for over 120 years. Here, we consider the synthesis and physico-chemical properties of the C-nitroso compounds and the available data regarding their biological activities. Synthetic methods reviewed include direct substitution of H by NO, oxidative approaches, and the addition of various oxides of nitrogen across multiple bonds. The electronic spectra of C-nitroso compounds and the mechanism and thermodynamics of monomer-dimer equilibration are described. The physico-chemical and biological properties of two related classes of compounds, the diazetine dioxides and the furoxans, are also described.

Authors
Gooden, DM; Chakrapani, H; Toone, EJ
MLA Citation
Gooden, DM, Chakrapani, H, and Toone, EJ. "C-nitroso compounds: synthesis, physicochemical properties and biological activities." Curr Top Med Chem 5.7 (2005): 687-705. (Review)
PMID
16101429
Source
pubmed
Published In
Current Topics in Medicinal Chemistry
Volume
5
Issue
7
Publish Date
2005
Start Page
687
End Page
705

Quantitative determination of analytes by force microscopy

Authors
Snyder, PW; Lee, G; Marszalke, PE; Clark, RL; Toone, EJ
MLA Citation
Snyder, PW, Lee, G, Marszalke, PE, Clark, RL, and Toone, EJ. "Quantitative determination of analytes by force microscopy (Submitted)." J. Am. Chem. Soc. (2005). (Academic Article)
Source
manual
Published In
J. Am. Chem. Soc.
Publish Date
2005

A droplet-based lab-on-a-chip for colorimetric detection of nitroaromatic explosives

Portable and automated field screening equipment would be very effective in detecting and quantifying explosives at various sites. A droplet-based microfluidic lab-on-a-chip utilizing electrowetting is presented for the colorimetric detection of TNT (trinitrotoluene). The method uses the reaction between nitroaromatics and a strong base which forms the highly colored Jackson-Meisenheimer complex. Microliter-sized droplets of TNT are programmed to transport, mix, and react with potassium hydroxide (KOH) on the microfluidic chip. Colorimetric reactions of TNT are characterized both on a spectrophotometer and on the microfluidic chip. The detection of TNT on the chip is linear in the range of 4-20μg/mL with a time-to-result of 2.5minutes. It is also observed that the absorbance peaks of DNT (dinitrotoluene) and TNT are mutually independent and that the presence of DNT does not affect the detection of TNT. Electrowetting also does not seem to influence the colorimetric complex as observed from a comparison of results between a spectrophotometer and on-chip. © 2005 IEEE.

Authors
Pamula, VK; Srinivasan, V; Chakrapani, H; Fair, RB; Toone, EJ
MLA Citation
Pamula, VK, Srinivasan, V, Chakrapani, H, Fair, RB, and Toone, EJ. "A droplet-based lab-on-a-chip for colorimetric detection of nitroaromatic explosives." Proceedings of the IEEE International Conference on Micro Electro Mechanical Systems (MEMS) (2005): 722-725.
Source
scival
Published In
Proceedings of the IEEE International Conference on Micro Electro Mechanical Systems (MEMS)
Publish Date
2005
Start Page
722
End Page
725
DOI
10.1109/MEMSYS.2005.1454031

A bacterial selection for the directed evolution of pyruvate aldolases.

A novel bacterial in vivo selection for pyruvate aldolase activity is described. Pyruvate kinase deficient cells, which lack the ability to biosynthetically generate pyruvate, require supplementation of exogenous pyruvate when grown on ribose. Supplementation with pyruvate concentrations as low as 50 microM rescues cell growth. A known substrate of the KDPG aldolases, 2-keto-4-hydroxy-4-(2'-pyridyl)butyrate (KHPB), also rescues cell growth, consistent with retroaldol cleavage by KDPG aldolase and rescue through pyruvate release. An initial round of selection against 2-keto-4-hydroxyoctonate (KHO), a nonsubstrate for wild-type aldolase, produced three mutants with intriguing alterations in protein sequence. This selection system allows rapid screening of mutant enzyme libraries and facilitates the discovery of enzymes with novel substrate specificities.

Authors
Griffiths, JS; Cheriyan, M; Corbell, JB; Pocivavsek, L; Fierke, CA; Toone, EJ
MLA Citation
Griffiths, JS, Cheriyan, M, Corbell, JB, Pocivavsek, L, Fierke, CA, and Toone, EJ. "A bacterial selection for the directed evolution of pyruvate aldolases." Bioorg Med Chem 12.15 (August 1, 2004): 4067-4074.
PMID
15246084
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry
Volume
12
Issue
15
Publish Date
2004
Start Page
4067
End Page
4074
DOI
10.1016/j.bmc.2004.05.034

Solid-phase synthesis for the identification of high-affinity bivalent lectin ligands.

The development of carbohydrate-based therapeutics has been frustrated by the low affinities that characterize protein-carbohydrate complexation. Because of the oligomeric nature of most lectins, the use of multivalency may offer a successful strategy for the creation of high-affinity ligands. The solid-phase evaluation of libraries of peptide-linked multivalent ligands facilitates rapid examination of a large fraction of linker structure space. If such solid-phase assays are to replicate solution binding behavior, the potential for intermolecular bivalent binding on bead surfaces must be eliminated. Here we report the solid-phase synthesis and analysis of peptide-linked, spatially segregated mono- and bivalent ligands for the legume lectin concanavalin A. Bead shaving protocols were used for the creation of beads displaying spatially segregated binding sequences on the surface of Tentagel resins. The same ligands were also synthesized on PEGA resin to determine the effect of ligand presentation on solid-phase binding. While we set out to determine the lower limit of assay sensitivity, the unexpected observation that intermolecular bivalent ligand binding is enhanced for bivalent ligands relative to monovalent ligands allowed direct observation of the level of surface blocking required to prevent intermolecular bivalent ligand binding. For a protein with binding sites separated by 65 A, approximately 99.9% of Tentagel(1) surface sites and 99.99% of the total sites on a PEGA bead must be blocked to prevent intermolecular bivalent binding. We also report agglutination and calorimetric solution-phase binding studies of mono- and bivalent peptide-linked ligands.

Authors
Debenham, SD; Snyder, PW; Toone, EJ
MLA Citation
Debenham, SD, Snyder, PW, and Toone, EJ. "Solid-phase synthesis for the identification of high-affinity bivalent lectin ligands." J Org Chem 68.15 (July 25, 2003): 5805-5811.
PMID
12868911
Source
pubmed
Published In
The Journal of Organic Chemistry
Volume
68
Issue
15
Publish Date
2003
Start Page
5805
End Page
5811
DOI
10.1021/jo0207271

Additivity and the physical basis of multivalency effects: a thermodynamic investigation of the calcium EDTA interaction.

To better understand the origin of multivalency effects in ligand binding, the binding of a series of mono-, bi-, tri- and tetravalent carboxylate ligands to Ca(II) was examined by isothermal titration calorimetry (ITC). The data are inconsistent with an entropic origin of enhanced affinity, but rather show that at least in this instance the multivalency effect is enthalpic in origin. Analysis of binding data using the Jencks model shows the addition of incremental carboxylate "ligands" produces an unfavorable interaction entropy that is more than offset by a strongly favorable interaction enthalpy. The most likely source of this interaction enthalpy is the relief of repulsive Coulombic interactions in the unbound state. The conformational entropy penalty arising from the restriction of flexible dihedrals is negligible, within experimental error. On the other hand, an enthalpic contribution from linker restriction contributes strongly to the overall thermodynamics of ligand binding. Together, these data suggest that enthalpic effects dominate ligand binding, and design strategies should seek to optimize these interactions. The incorporation of unfavorable interactions in the unbound ligand that are relieved during binding provides an important mechanism by which to enhance ligand affinities.

Authors
Christensen, T; Gooden, DM; Kung, JE; Toone, EJ
MLA Citation
Christensen, T, Gooden, DM, Kung, JE, and Toone, EJ. "Additivity and the physical basis of multivalency effects: a thermodynamic investigation of the calcium EDTA interaction." J Am Chem Soc 125.24 (June 18, 2003): 7357-7366.
PMID
12797810
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
125
Issue
24
Publish Date
2003
Start Page
7357
End Page
7366
DOI
10.1021/ja021240c

Nitroxyl disulfides, novel intermediates in transnitrosation reactions.

A novel anionic RSN(O)SR species, the intermediate in transnitrosation reactions, was explored computationally with B3LYP and CBS-QB3 methods. The species resembles a nitroxyl coordinated to a highly distorted disulfide, and it differs significantly from intermediates in nucleophilic acyl substitution. Reactions of the following species were computed for comparison: MeS(-) + MeSNO; MeO(-) + MeONO; MeS(-) + MeSCHO; MeO(-) + MeOCHO. The last two have very different intermediates from the first two. Mass spectrometric experimental evidence is presented that is consistent with the formation of a nitroxyl disulfide in the gas phase. The calculated proton affinity and redox potentials of the intermediate are also reported.

Authors
Houk, KN; Hietbrink, BN; Bartberger, MD; McCarren, PR; Choi, BY; Voyksner, RD; Stamler, JS; Toone, EJ
MLA Citation
Houk, KN, Hietbrink, BN, Bartberger, MD, McCarren, PR, Choi, BY, Voyksner, RD, Stamler, JS, and Toone, EJ. "Nitroxyl disulfides, novel intermediates in transnitrosation reactions." J Am Chem Soc 125.23 (June 11, 2003): 6972-6976.
PMID
12783550
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
125
Issue
23
Publish Date
2003
Start Page
6972
End Page
6976
DOI
10.1021/ja029655l

Binding of ochratoxin a to human serum albumin stabilized by a protein-ligand ion pair

Ochratoxin A (OTA), a fungal metabolite of strains of Penicillium and Aspergillus, binds in its dianion form to Sudlow site I of human serum albumin (HSA) with high affinity. In this study, isothermal calorimetry (ITC) is used to study the binding of OTA and its O-methyl derivative (MOA). Calculations of the equilibrium geometry of the monoanion and dianion of OTA reveal only small structural changes among the lowest energy conformers. The ITC data show the binding of MOA, which lacks the phenolic proton of OTA, is accompanied by the uptake of a proton from the surrounding solvent. At pH 7.13, the binding of OTA is accompanied by uptake of 0.43 ± 0.15 protons from the solvent. At this pH, the monoanion (0.54) and dianion (0.46) forms of OTA are both present in solution. However, the pKa of the phenolic group of OTA decreases by more than three units upon protein binding, and so all available OTA is bound to the protein as the dianion. To account for the ITC data, a model is proposed in which the proton is provided by the phenolic moiety of OTA in the case of initial binding of the monoanion, and a proton is taken up from the surrounding solvent for initial binding of the dianion. The binding constant of MOA is 2 orders of magnitude smaller than that of OTA, indicating the ion pair between the phenoxide group of OTA and the protonated amino acid is a major contributor to the high binding affinity of OTA to HSA. To identify the specific amino acid involved, the binding of OTA to bovine, rat, and porcine serum albumins was examined. Deprotonation of the monoanion of OTA occurred upon binding to all species. Assuming the amino acid is conserved between species and taking into account crystal structures of ligands bound to site I of HSA and their ability to displace OTA from HSA, either R218 or R257 is involved in the ion pairing with OTA. These two amino acids sit across the binding cavity from one other in site I.

Authors
Perry, JL; Christensen, T; Goldsmith, MR; Toone, EJ; Beratan, DN; Simon, JD
MLA Citation
Perry, JL, Christensen, T, Goldsmith, MR, Toone, EJ, Beratan, DN, and Simon, JD. "Binding of ochratoxin a to human serum albumin stabilized by a protein-ligand ion pair." Journal of Physical Chemistry B 107.31 (2003): 7884-7888.
Source
scival
Published In
Journal of Physical Chemistry B
Volume
107
Issue
31
Publish Date
2003
Start Page
7884
End Page
7888

Calorimetric evaluation of protein-carbohydrate affinities.

Authors
Christensen, T; Toone, EJ
MLA Citation
Christensen, T, and Toone, EJ. "Calorimetric evaluation of protein-carbohydrate affinities." Methods Enzymol 362 (2003): 486-504.
PMID
12968383
Source
pubmed
Published In
Methods in Enzymology
Volume
362
Publish Date
2003
Start Page
486
End Page
504
DOI
10.1016/S0076-6879(03)01032-2

The decomposition of thionitrites.

The mechanism of thionitrite decomposition, both in vivo and in vitro, remains unclear. Thionitrite stability is highly variable; it is a complex function of thionitrite structure and environmental condition. Several recent advances clarify the role of unimolecular homlytic decomposition, metal-catalyzed reductive decomposition and higher-order enzymatic and non-enzymatic processes to the overall observed stability of thionitrites.

Authors
Stamler, JS; Toone, EJ
MLA Citation
Stamler, JS, and Toone, EJ. "The decomposition of thionitrites." Curr Opin Chem Biol 6.6 (December 2002): 779-785. (Review)
PMID
12470731
Source
pubmed
Published In
Current Opinion in Chemical Biology
Volume
6
Issue
6
Publish Date
2002
Start Page
779
End Page
785

Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase.

The Thermotoga maritima aldolase gene has been cloned into a T7 expression vector and overexpressed in Escherichia coli. The preparation yields 470 UL(-1) of enzyme at a specific activity of 9.4 U mg(-1). During retroaldol cleavage of KDPG, the enzyme shows a k(cat) that decreases with decreasing temperature. A more than offsetting decrease in K(m) yields an enzyme that is more efficient at 40 degrees C than at 70 degrees C. The substrate specificity of the enzyme was evaluated in the synthetic direction with a range of aldehyde substrates. Although the protein shows considerable structural homology to KDPG aldolases from mesophilic sources, significant differences in substrate specificity exist. A preparative scale reaction between 2-pyridine carboxaldehyde and pyruvate provided product of the same absolute configuration as mesophilic enzymes, but with diminished stereoselectivity.

Authors
Griffiths, JS; Wymer, NJ; Njolito, E; Niranjanakumari, S; Fierke, CA; Toone, EJ
MLA Citation
Griffiths, JS, Wymer, NJ, Njolito, E, Niranjanakumari, S, Fierke, CA, and Toone, EJ. "Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase." Bioorg Med Chem 10.3 (March 2002): 545-550.
PMID
11814840
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry
Volume
10
Issue
3
Publish Date
2002
Start Page
545
End Page
550

The cluster glycoside effect.

Authors
Lundquist, JJ; Toone, EJ
MLA Citation
Lundquist, JJ, and Toone, EJ. "The cluster glycoside effect." Chem Rev 102.2 (February 2002): 555-578. (Review)
PMID
11841254
Source
pubmed
Published In
Chemical Reviews
Volume
102
Issue
2
Publish Date
2002
Start Page
555
End Page
578

Towards high affinity carbohydrate-binding proteins: Directed evolution of murine galectin-3

Towards a better understanding of the molecular basis of affinity, a directed evolution of murine galectin-3 (G3) was initiated to produce mutants with improved affinity for lactose and N-acetyllactosamine relative to the wild-type protein. A series of N-terminal truncations were developed to facilitate incorporation of the 35 kDa protein into a phage-display construct. Analysis of the various assemblies revealed that all such deletions produced protein unsuitable for use in directed evolution studies. Following fusion of the full-length galectin to p3 of filamentous phage, three libraries were constructed and biopanned for increased affinity for lactose. The first two libraries, of 1 × 105 and 1 × 106 members, respectively, were assembled through a combination of error-prone PCR and DNA shuffling. A third library was constructed using a modified staggered extension protocol (StEP), but contained only 10 members. Mutants were also engineered site-specifically to test the role of key residues in or near the binding pocket. Analysis of the mutants by ITC identified one mutation (R158G) that produces a twofold increase in affinity for lactose and another that results in a sixfold increase in affinity for N-acetyllactosamine. Solid-phase binding analysis of phage for nonexpressing proteins indicated that two other mutants demonstrated increased binding to β-methyllactose relative to the wild-type protein. Together these studies validate the evolutionary approach and set the stage for the development of novel carbohydrate-binding proteins.

Authors
Lundquist, JJ; Kiburz, BM; Wu, JK; Jr, KDG; Toone, EJ
MLA Citation
Lundquist, JJ, Kiburz, BM, Wu, JK, Jr, KDG, and Toone, EJ. "Towards high affinity carbohydrate-binding proteins: Directed evolution of murine galectin-3." Canadian Journal of Chemistry 80.8 (2002): 999-1009.
Source
scival
Published In
Canadian Journal of Chemistry
Volume
80
Issue
8
Publish Date
2002
Start Page
999
End Page
1009
DOI
10.1139/v02-086

Analysis of S-nitrosothiol decomposition

The fate of various low molecular weight RSNO compounds under different conditions were studied by employing an Agilent 1100 LC/MCD ion trap. Electrospray ionization (ESI) was utilized and ion were detected in the negative ion mode with the expectation of S-nitroso-L-cysteine ethyl ester hydrochlorides. Scans were obtained either by direct infusion of following ion isolation inside the trap for to 8 seconds. The total ion spectrum of SNAP in acetonitrile shows ions belonging to that of the RSNO, the RSNO dimer and the RSSR disulfides.

Authors
Choi, BY; Voyksner, RD; Toone, EJ
MLA Citation
Choi, BY, Voyksner, RD, and Toone, EJ. "Analysis of S-nitrosothiol decomposition." Proceedings 50th ASMS Conference on Mass Spectrometry and Allied Topics (2002): 799-800.
Source
scival
Published In
Proceedings 50th ASMS Conference on Mass Spectrometry and Allied Topics
Publish Date
2002
Start Page
799
End Page
800

S-N dissociation energies of S-nitrosothiols: on the origins of nitrosothiol decomposition rates.

Authors
Bartberger, MD; Mannion, JD; Powell, SC; Stamler, JS; Houk, KN; Toone, EJ
MLA Citation
Bartberger, MD, Mannion, JD, Powell, SC, Stamler, JS, Houk, KN, and Toone, EJ. "S-N dissociation energies of S-nitrosothiols: on the origins of nitrosothiol decomposition rates." J Am Chem Soc 123.36 (September 12, 2001): 8868-8869.
PMID
11535101
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
123
Issue
36
Publish Date
2001
Start Page
8868
End Page
8869

Thermodynamics of metal ion binding. 1. Metal ion binding by wild-type carbonic anhydrase.

Understanding the energetic consequences of molecular structure in aqueous solution is a prerequisite to the rational design of synthetic motifs with predictable properties. Such properties include ligand binding and the collapse of polymer chains into discrete three-dimensional structures. Despite advances in macromolecular structure determination, correlations of structure with high-resolution thermodynamic data remain limited. Here we compare thermodynamic parameters for the binding of Zn(II), Cu(II), and Co(II) to human carbonic anhydrase II. These calorimetrically determined values are interpreted in terms of high-resolution X-ray crystallographic data. While both zinc and cobalt are bound with a 1:1 stoichiometry, CAII binds two copper ions. Considering only the high-affinity site, there is a diminution in the enthalpy of binding through the series Co(II) --> Zn(II) --> Cu(II) that mirrors the enthalpy of hydration; this observation reinforces the notion that the thermodynamics of solute association with water is at least as important as the thermodynamics of solute-solute interaction and that these effects must be considered when interpreting association in aqueous solution. Additionally, DeltaC(p) data suggest that zinc binding to CAII proceeds with a greater contribution from desolvation than does binding of either copper or cobalt, suggesting Nature optimizes binding by optimizing desolvation.

Authors
DiTusa, CA; Christensen, T; McCall, KA; Fierke, CA; Toone, EJ
MLA Citation
DiTusa, CA, Christensen, T, McCall, KA, Fierke, CA, and Toone, EJ. "Thermodynamics of metal ion binding. 1. Metal ion binding by wild-type carbonic anhydrase." Biochemistry 40.18 (May 8, 2001): 5338-5344.
PMID
11330996
Source
pubmed
Published In
Biochemistry
Volume
40
Issue
18
Publish Date
2001
Start Page
5338
End Page
5344

S-nitrosothiol repletion by an inhaled gas regulates pulmonary function.

NO synthases are widely distributed in the lung and are extensively involved in the control of airway and vascular homeostasis. It is recognized, however, that the O(2)-rich environment of the lung may predispose NO toward toxicity. These Janus faces of NO are manifest in recent clinical trials with inhaled NO gas, which has shown therapeutic benefit in some patient populations but increased morbidity in others. In the airways and circulation of humans, most NO bioactivity is packaged in the form of S-nitrosothiols (SNOs), which are relatively resistant to toxic reactions with O(2)/O(2)(-). This finding has led to the proposition that channeling of NO into SNOs may provide a natural defense against lung toxicity. The means to selectively manipulate the SNO pool, however, has not been previously possible. Here we report on a gas, O-nitrosoethanol (ENO), which does not react with O(2) or release NO and which markedly increases the concentration of indigenous species of SNO within airway lining fluid. Inhalation of ENO provided immediate relief from hypoxic pulmonary vasoconstriction without affecting systemic hemodynamics. Further, in a porcine model of lung injury, there was no rebound in cardiopulmonary hemodynamics or fall in oxygenation on stopping the drug (as seen with NO gas), and additionally ENO protected against a decline in cardiac output. Our data suggest that SNOs within the lung serve in matching ventilation to perfusion, and can be manipulated for therapeutic gain. Thus, ENO may be of particular benefit to patients with pulmonary hypertension, hypoxemia, and/or right heart failure, and may offer a new therapeutic approach in disorders such as asthma and cystic fibrosis, where the airways may be depleted of SNOs.

Authors
Moya, MP; Gow, AJ; McMahon, TJ; Toone, EJ; Cheifetz, IM; Goldberg, RN; Stamler, JS
MLA Citation
Moya, MP, Gow, AJ, McMahon, TJ, Toone, EJ, Cheifetz, IM, Goldberg, RN, and Stamler, JS. "S-nitrosothiol repletion by an inhaled gas regulates pulmonary function." Proc Natl Acad Sci U S A 98.10 (May 8, 2001): 5792-5797.
PMID
11320213
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
10
Publish Date
2001
Start Page
5792
End Page
5797
DOI
10.1073/pnas.091109498

Thermodynamics of metal ion binding. 2. Metal ion binding by carbonic anhydrase variants.

The ability to construct molecular motifs with predictable properties in aqueous solution requires an extensive knowledge of the relationships between structure and energetics. The design of metal binding motifs is currently an area of intense interest in the bioorganic community. To date synthetic motifs designed to bind metal ions lack the remarkable affinities observed in biological systems. To better understand the structural basis of metal ion affinity, we report here the thermodynamics of binding of divalent zinc ions to wild-type and mutant carbonic anhydrases and the interpretation of these parameters in terms of structure. Mutations were made both to the direct His ligand at position 94 and to indirect, or second-shell, ligands Gln-92, Glu-117, and Thr-199. The thermodynamics of ligand binding by several mutant proteins is complicated by the development of a second zinc binding site on mutation; such effects must be considered carefully in the interpretation of thermodynamic data. In all instances modification of the protein produces a complex series of changes in both the enthalpy and entropy of ligand binding. In most cases these effects are most readily rationalized in terms of ligand and protein desolvation, rather than in terms of changes in the direct interactions of ligand and protein. Alteration of second-shell ligands, thought to function primarily by orienting the direct ligands, produces profoundly different effects on the enthalpy of binding, depending on the nature of the residue. These results suggest a range of activities for these ligands, contributing both enthalpic and entropic effects to the overall thermodynamics of binding. Together, our results demonstrate the importance of understanding relationships between structure and hydration in the construction of novel ligands and biological polymers.

Authors
DiTusa, CA; McCall, KA; Christensen, T; Mahapatro, M; Fierke, CA; Toone, EJ
MLA Citation
DiTusa, CA, McCall, KA, Christensen, T, Mahapatro, M, Fierke, CA, and Toone, EJ. "Thermodynamics of metal ion binding. 2. Metal ion binding by carbonic anhydrase variants." Biochemistry 40.18 (May 8, 2001): 5345-5351.
PMID
11330997
Source
pubmed
Published In
Biochemistry
Volume
40
Issue
18
Publish Date
2001
Start Page
5345
End Page
5351

Directed evolution of a new catalytic site in 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli.

BACKGROUND: Aldolases are carbon bond-forming enzymes that have long been identified as useful tools for the organic chemist. However, their utility is limited in part by their narrow substrate utilization. Site-directed mutagenesis of various enzymes to alter their specificity has been performed for many years, typically without the desired effect. More recently directed evolution has been employed to engineer new activities onto existing scaffoldings. This approach allows random mutation of the gene and then selects for fitness to purpose those proteins with the desired activity. To date such approaches have furnished novel activities through multiple mutations of residues involved in recognition; in no instance has a key catalytic residue been altered while activity is retained. RESULTS: We report a double mutant of E. coli 2-keto-3-deoxy-6-phosphogluconate aldolase with reduced but measurable enzyme activity and a synthetically useful substrate profile. The mutant was identified from directed-evolution experiments. Modification of substrate specificity is achieved by altering the position of the active site lysine from one beta strand to a neighboring strand rather than by modification of the substrate recognition site. The new enzyme is different to all other existing aldolases with respect to the location of its active site to secondary structure. The new enzyme still displays enantiofacial discrimination during aldol addition. We have determined the crystal structure of the wild-type enzyme (by multiple wavelength methods) to 2.17 A and the double mutant enzyme to 2.7 A resolution. CONCLUSIONS: These results suggest that the scope of directed evolution is substantially larger than previously envisioned in that it is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. The structure of the double mutant shows how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate.

Authors
Wymer, N; Buchanan, LV; Henderson, D; Mehta, N; Botting, CH; Pocivavsek, L; Fierke, CA; Toone, EJ; Naismith, JH
MLA Citation
Wymer, N, Buchanan, LV, Henderson, D, Mehta, N, Botting, CH, Pocivavsek, L, Fierke, CA, Toone, EJ, and Naismith, JH. "Directed evolution of a new catalytic site in 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli." Structure 9.1 (January 10, 2001): 1-9.
PMID
11342129
Source
pubmed
Published In
Structure
Volume
9
Issue
1
Publish Date
2001
Start Page
1
End Page
9

Thermodynamics of Metal Ion Bonding. II. Metal Ion Binding by Mutated Carbonic Anhydrases.

Authors
Ditusa, CA; Fierke, CA; Toone, EJ
MLA Citation
Ditusa, CA, Fierke, CA, and Toone, EJ. "Thermodynamics of Metal Ion Bonding. II. Metal Ion Binding by Mutated Carbonic Anhydrases." Biochemistry 40.18 (2001): 5345-5351. (Academic Article)
Source
manual
Published In
Biochemistry
Volume
40
Issue
18
Publish Date
2001
Start Page
5345
End Page
5351

Multivalency effects in protein--carbohydrate interaction: the binding of the Shiga-like toxin 1 binding subunit to multivalent C-linked glycopeptides.

A series of monovalent and bivalent glycopeptides displaying a C-linked analogue of the Pk trisaccharide, the in vivo ligand for the pentavalent Shiga-like toxin binding subunit (SLT-1B), were prepared and evaluated as ligands for SLT-1B by isothermal titration microcalorimetry and competitive enzyme-linked immunosorbent assay (ELISA). Although none of the monovalent ligands showed any enhancement in affinity compared to O-methyl glycoside, two bivalent ligands show significant enhancements in affinity in assays. This observation represents the first calorimetric observation of an enhancement in affinity for this system. In contrast, only one of the two ligands shows an enhancement in the competitive ELISA. Together, these data signal a difference in the means by which the two ligands achieve affinity, apparently triggered by a change in the nature of the linker domain. These results provide a rationalization for apparently contradictory reports from the recent literature and again emphasize the importance of investigating complex binding phenomena by multiple techniques.

Authors
Lundquist, JJ; Debenham, SD; Toone, EJ
MLA Citation
Lundquist, JJ, Debenham, SD, and Toone, EJ. "Multivalency effects in protein--carbohydrate interaction: the binding of the Shiga-like toxin 1 binding subunit to multivalent C-linked glycopeptides." J Org Chem 65.24 (December 1, 2000): 8245-8250.
PMID
11101380
Source
pubmed
Published In
The Journal of Organic Chemistry
Volume
65
Issue
24
Publish Date
2000
Start Page
8245
End Page
8250

Microcalorimetric determination of thermodynamic parameters for ionophore-siderophore host-guest complex formation.

Thermodynamic parameters (delta H, delta S, and delta G) were determined by microcalorimetry in wet chloroform for host-guest assembly formation involving second-sphere complexation of the siderophore ferrioxamine B by crown ether (18-crown-6, cis-dicyclohexano-18-crown-6, benzo-18-crown-6) and cryptand (2.2.2 cryptand) hosts. Similar data were also collected for the same hosts with the pentylammonium ion guest, which corresponds to the pendant pentylamine side chain of ferroxamine B. Host-guest assembly formation constants (Ka) obtained from microcalorimetry agree with values obtained indirectly from chloroform/water extraction studies in those cases where comparable data are available. On the basis of a trend established by the pentylammonium guest, an enhanced stability relative to the crown ethers is observed for the assembly composed of ferrioxamine B and 2.2.2 cryptand that is due to entropic effects. Trends in delta H and delta S with changes in host and guest structure are discussed and attributed directly to host-guest complex formation, as solvation effects were determined to be insignificant (delta Cp = 0).

Authors
Trzaska, SM; Toone, EJ; Crumbliss, AL
MLA Citation
Trzaska, SM, Toone, EJ, and Crumbliss, AL. "Microcalorimetric determination of thermodynamic parameters for ionophore-siderophore host-guest complex formation." Inorg Chem 39.6 (March 20, 2000): 1071-1075.
PMID
12526393
Source
pubmed
Published In
Inorganic Chemistry
Volume
39
Issue
6
Publish Date
2000
Start Page
1071
End Page
1075

Enzyme-catalyzed synthesis of carbohydrates.

Several new enzymes of utility in the synthesis of carbohydrates have been reported during the past year. Additionally, the utility of several well studied enzymes has been expanded. Pyruvate aldolases, aldolase abzymes and both wild-type and mutated glycosidases have found increasing acceptance in the community. Preliminary reports suggest that thermophilic enzymes may possess significant advantages compared to their mesophilic counterparts for carbohydrate synthesis.

Authors
Wymer, N; Toone, EJ
MLA Citation
Wymer, N, and Toone, EJ. "Enzyme-catalyzed synthesis of carbohydrates." Curr Opin Chem Biol 4.1 (February 2000): 110-119. (Review)
PMID
10679369
Source
pubmed
Published In
Current Opinion in Chemical Biology
Volume
4
Issue
1
Publish Date
2000
Start Page
110
End Page
119

A Facile Method for the Reductive Deprotection of Carbohydrate Benzylidene Protecting Groups.

Authors
Debenham, S; Toone, EJ
MLA Citation
Debenham, S, and Toone, EJ. "A Facile Method for the Reductive Deprotection of Carbohydrate Benzylidene Protecting Groups." Tetrahedron Asymm. (2000): 385-387. (Academic Article)
Source
manual
Published In
Tetrahedron Asymm.
Publish Date
2000
Start Page
385
End Page
387

Biological and Calorimetric Analysis of Multivalent Glycodendrimers Ligands for Concanavalin A.

Authors
Corbell, JB; Lundquist, JJ; Toone, EJ
MLA Citation
Corbell, JB, Lundquist, JJ, and Toone, EJ. "Biological and Calorimetric Analysis of Multivalent Glycodendrimers Ligands for Concanavalin A." Tetrahedron Asymm. 11 (2000): 95-111. (Academic Article)
Source
manual
Published In
Tetrahedron Asymm.
Volume
11
Publish Date
2000
Start Page
95
End Page
111

Calorimetric and Biological Evaluation of Shigalike Toxin1 Binding to Multivalent C-Linked Glycopeptides

Authors
Lundquist, JJ; Debenham, SD; Toone, EJ
MLA Citation
Lundquist, JJ, Debenham, SD, and Toone, EJ. "Calorimetric and Biological Evaluation of Shigalike Toxin1 Binding to Multivalent C-Linked Glycopeptides." J. Org. Chem. 65 (2000): 8245-8250. (Academic Article)
Source
manual
Published In
J. Org. Chem.
Volume
65
Publish Date
2000
Start Page
8245
End Page
8250

Enzymatic Carbohydrate Synthesis

Authors
Wymer, N; Toone, EJ
MLA Citation
Wymer, N, and Toone, EJ. "Enzymatic Carbohydrate Synthesis." Current Opinion in Chemical Biology 4 (2000): 110-119. (Academic Article)
Source
manual
Published In
Current Opinion in Chemical Biology
Volume
4
Publish Date
2000
Start Page
110
End Page
119

Nuclear Magnetic Resonance and Visible Spectroscopy of S-Nitrosothiols. Conformational Distribution Dictates Spectroscopic Behavior.

Authors
Bartberger, MD; Houk, KN; Powell, S; Mannion, J; Lo, KYK; Stamler, JS; Toone, EJ
MLA Citation
Bartberger, MD, Houk, KN, Powell, S, Mannion, J, Lo, KYK, Stamler, JS, and Toone, EJ. "Nuclear Magnetic Resonance and Visible Spectroscopy of S-Nitrosothiols. Conformational Distribution Dictates Spectroscopic Behavior." J. Am. Chem. Soc. 122 (2000): 5889-5890. (Academic Article)
Source
manual
Published In
J. Am. Chem. Soc.
Volume
122
Publish Date
2000
Start Page
5889
End Page
5890

Theory, spectroscopy, and crystallographic analysis of S-nitrosothiols: Conformational distribution dictates spectroscopic behavior [7]

Authors
Bartberger, MD; Houk, KN; Powell, SC; Mannion, JD; Lo, KY; Stamler, JS; Toone, EJ
MLA Citation
Bartberger, MD, Houk, KN, Powell, SC, Mannion, JD, Lo, KY, Stamler, JS, and Toone, EJ. "Theory, spectroscopy, and crystallographic analysis of S-nitrosothiols: Conformational distribution dictates spectroscopic behavior [7]." Journal of the American Chemical Society 122.24 (2000): 5889-5890.
Source
scival
Published In
Journal of the American Chemical Society
Volume
122
Issue
24
Publish Date
2000
Start Page
5889
End Page
5890
DOI
10.1021/ja994476y

Regioselective reduction of 4,6-O-benzylidenes using triethylsilane and BF3.Et2O

The 4,6-di-O-benzylidene acetals of glucose, mannose, glucosamine, and galactose were regioselectively reduced by triethylsilane in the presence of BF3.Et2O to yield the 6-O-benzyl ethers in good to excellent yields. Copyright (C) 2000 Elsevier Science Ltd.

Authors
Debenham, SD; Toone, EJ
MLA Citation
Debenham, SD, and Toone, EJ. "Regioselective reduction of 4,6-O-benzylidenes using triethylsilane and BF3.Et2O." Tetrahedron Asymmetry 11.2 (2000): 385-387.
Source
scival
Published In
Tetrahedron Asymmetry
Volume
11
Issue
2
Publish Date
2000
Start Page
385
End Page
387
DOI
10.1016/S0957-4166(99)00584-4

A comparison of biological and calorimetric analyses of multivalent glycodendrimer ligands for concanavalin A

The cluster glycoside effect - the observation that multivalent glycosides bind to their polyvalent protein receptors with apparent affinities greater than those that can be rationalized solely on the basis of valency - is by now a well established phenomenon. As part of a continuing effort to provide a molecular basis for the cluster glycoside effect, we report here the synthesis of two series of mannosylated dendritic ligands and their performance in a range of competitive and non-competitive binding assays, including hemeagglutination inhibition (HIA), enzyme-linked lectin assays (ELLA) and isothermal titration microcalorimetry (ITC). The first series of ligands contained a semi-rigid glycylglycine spacer and showed no significant performance enhancement in any binding studies. The second series of ligands contained a flexible tetraethylene glycol spacer; these ligands showed marked enhancements at tetravalent and hexavalent levels in both HIA (IC50=3 and<0.8 μM, respectively) and ITC (K(A)=6.2x104 and 1.5x106 M-1, respectively) studies. In all cases, the thermodynamic parameters of association are consistent with non-specific aggregation rather than enhanced lectin-ligand affinity. This conclusion is reinforced by the lack of enhancements in ligand activity observed in ELLA studies. Copyright (C) 2000 Elsevier Science Ltd.

Authors
Corbell, JB; Lundquist, JJ; Toone, EJ
MLA Citation
Corbell, JB, Lundquist, JJ, and Toone, EJ. "A comparison of biological and calorimetric analyses of multivalent glycodendrimer ligands for concanavalin A." Tetrahedron Asymmetry 11.1 (2000): 95-111.
Source
scival
Published In
Tetrahedron Asymmetry
Volume
11
Issue
1
Publish Date
2000
Start Page
95
End Page
111
DOI
10.1016/S0957-4166(99)00589-3

Initiating a Structural Study of KDPG Aldolase from Escherichia coli

Authors
Wymer, N; Buchanan, LV; Mehta, N; Pocivavsek, L; Nirjanakumari, S; Toone, EJ; Naismith, JH
MLA Citation
Wymer, N, Buchanan, LV, Mehta, N, Pocivavsek, L, Nirjanakumari, S, Toone, EJ, and Naismith, JH. "Initiating a Structural Study of KDPG Aldolase from Escherichia coli." Acta Cryst. D55 (1999): 1946-1948. (Academic Article)
Source
manual
Published In
Acta Cryst.
Volume
D55
Publish Date
1999
Start Page
1946
End Page
1948

On the meaning of affinity: Cluster glycoside effects and concanavalin A

The inhibition of protein-carbohydrate interaction provides a powerful therapeutic strategy for the treatment of myriad human diseases. To date, application of such approaches have been frustrated by the inherent low affinity of carbohydrate ligands for their protein receptors. Because lectins typically exist in multimeric assemblies, a variety of polyvalent saccharide ligands have been prepared in the search for high affinity. The cluster glycoside effect, or the observation of high affinity derived from multivalency in oligosaccharide ligands, apparently represents the best strategy for overcoming the 'weak binding' problem. Here we report the synthesis of a series of multivalent dendritic saccharides and a biophysical evaluation of their interaction with the plant lectin concanavalin A. Although a 30-fold enhancement in affinity on a valence-corrected basis is observed by agglutination assay, calorimetric titration of soluble protein with a range of multivalent ligands reveals no enhancement in binding free energies. Rather, IC50 values from agglutination measurements correlate well with entropies of binding. This observation suggests that hemagglutination measures a phenomenon distinct from binding that is typified by a large favorable entropy and an unfavorable enthalpy: this process is almost certainly aggregation. Supporting this assertion, we report crystal structures of multivalent ligands cross-linking concanavalin A dimers. To the best of our knowledge, these structures are the first reported of their kind. Our results indicate that hemagglutination assays evaluate the ability of ligands to inhibit the formation of cross-linked lattices, a process only tangentially related to reversible ligand binding. Cluster glycoside effects observed in agglutination assays must, therefore, be viewed with caution. Such effects may or may not be relevant to the design of therapeutically useful saccharides.

Authors
Dimick, SM; Powell, SC; McMahon, SA; Moothoo, DN; Naismith, JH; Toone, EJ
MLA Citation
Dimick, SM, Powell, SC, McMahon, SA, Moothoo, DN, Naismith, JH, and Toone, EJ. "On the meaning of affinity: Cluster glycoside effects and concanavalin A." Journal of the American Chemical Society 121.44 (1999): 10286-10296.
Source
scival
Published In
Journal of the American Chemical Society
Volume
121
Issue
44
Publish Date
1999
Start Page
10286
End Page
10296
DOI
10.1021/ja991729e

Synthesis of α- and β-carbon-linked serine analogues of the P(k) trisaccharide

The synthesis of glycopeptide ligands for a range of biomedically relevant carbohydrate-binding proteins is a topic of great importance to the glycobiology community. This task is impeded by the inherent instability of glycosyl linkages to serine/threonine, the normal sites of O-glycosylation in proteins. We have previously developed methodology for the preparation of C- glycosylated serines based on catalytic asymmetric hydrogenation of the corresponding enamide esters with the DuPHOS-Rh+ catalysts. Here we report further development of the methodology in the preparation of the C-glycosyl serine analogue of the pk trisaccharide (α-Gal(1→4)β-Gal(1→4)β-Glc-CH2- serine); we require these ligands for our continuing investigations of the binding subunit of the shiga-like toxin. Catalytic asymmetric hydrogenation was used to prepare both α- and β-C-glycosides in the R and S serine series. We report here on the tolerance of the DuPHOS catalysts toward acetate, benzoate, and benzyl hydroxyl protecting groups. Additionally, we have developed an amino acid protecting group strategy compatible with both asymmetric hydrogenation and solid-phase peptide synthesis. In the course of our studies, we have also developed a new methodology for regioselective reductive cleavage of benzylidene protecting groups.

Authors
Debenham, SD; Cossrow, J; Toone, EJ
MLA Citation
Debenham, SD, Cossrow, J, and Toone, EJ. "Synthesis of α- and β-carbon-linked serine analogues of the P(k) trisaccharide." Journal of Organic Chemistry 64.25 (1999): 9153-9163.
Source
scival
Published In
Journal of Organic Chemistry
Volume
64
Issue
25
Publish Date
1999
Start Page
9153
End Page
9163
DOI
10.1021/jo991096m

Initiating a structural study of 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli

2-Keto-3-deoxy-6-phosphogluconate aldolase (KDPG aldolase, E.C. 4.1.2.14) is a member of the pyruvate/phosphoenolpyruvate aldolase family. It is also a synthetically useful enzyme, capable of catalyzing the stereoselective aldol addition of pyruvate to a range of unnatural electrophilic substrates. The recombinant protein was purified by a two-step HPLC protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated the protein to be monodisperse. Crystals were obtained using the sitting-drop vapour-diffusion method, with PEG 6K as precipitant. Diffraction data were collected on a frozen crystal to a resolution of 2.26 Å on station PX9.6 at the Daresbury synchrotron. The crystal belongs to space group P212121, with unit-cell parameters a = 53.2, b = 77.9, c = 146.8 Å.

Authors
Buchanan, LV; Mehta, N; Pocivavsek, L; Niranjanakumari, S; Toone, EJ; Naismith, JH
MLA Citation
Buchanan, LV, Mehta, N, Pocivavsek, L, Niranjanakumari, S, Toone, EJ, and Naismith, JH. "Initiating a structural study of 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli." Acta Crystallographica Section D: Biological Crystallography 55.11 (1999): 1946-1948.
Source
scival
Published In
Acta Crystallographica Section D
Volume
55
Issue
11
Publish Date
1999
Start Page
1946
End Page
1948
DOI
10.1107/S0907444999011166

Crystallization of succinylated concanavalin A bound to a synthetic bivalent ligand and preliminary structural analysis.

Crystals have been obtained of succinylated concanavalin A complexed to a novel bidentate synthetic ligand. The crystals are the first example of a lectin with a synthetic multivalent ligand and the first report of crystallization of succinylated concanavalin A. The crystals were obtained by sitting-drop vapour diffusion equilibrating with a solution of 20% polyethylene glycol, pH 5, 293. 5 K. Crystals are orthorhombic, belonging to space group C2221 with unit-cell dimensions of a = 99.1, b = 127.4, c = 118.9 A. The asymmetric unit contains a dimer, with over 65% of the volume occupied by water. The ligand cross links concanavalin A monomers. Succinylated concanavalin A is known to be a dimer in solution, yet it is found as the typical concanavalin A tetramer in the crystal. The contacts holding together the tetramer appear extensive and suggest that a fine balance between dimer and tetramers exists. Data to 2.65 A have been collected and the structure determined by the molecular replacement method.

Authors
Moothoo, DN; McMahon, SA; Dimick, SM; Toone, EJ; Naismith, JH
MLA Citation
Moothoo, DN, McMahon, SA, Dimick, SM, Toone, EJ, and Naismith, JH. "Crystallization of succinylated concanavalin A bound to a synthetic bivalent ligand and preliminary structural analysis." Acta Crystallogr D Biol Crystallogr 54.Pt 5 (September 1, 1998): 1023-1025.
PMID
9757127
Source
pubmed
Published In
Acta Crystallographica Section D
Volume
54
Issue
Pt 5
Publish Date
1998
Start Page
1023
End Page
1025

The synthetic utility of KDPGal aldolase

We describe the isolation and purification of KDPGal aldolase from two sources. To facilitate this isolation, a facile five-step chemical synthesis of 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) (1) from commercially available D-galactono-1,4-lactone (3) is described. The key steps are the β- elimination of the protected aldonolactone 4 to furnish the desired 2,3- unsaturated ketone 5 and selective phosphorylation of the primary hydroxyl moiety of the diol 7. The purity of the KDPGal sample was > 95% as determined by enzymatic assay using crude KDPGal aldolase from Pseudomonas saccharophila. An initial substrate specificity and pH-activity profile of KDPGal aldolase are described.

Authors
Cotterill, IC; Henderson, DP; Shelton, MC; Toone, EJ
MLA Citation
Cotterill, IC, Henderson, DP, Shelton, MC, and Toone, EJ. "The synthetic utility of KDPGal aldolase." Journal of Molecular Catalysis - B Enzymatic 5.1-4 (1998): 103-111.
Source
scival
Published In
Journal of Molecular Catalysis - B Enzymatic
Volume
5
Issue
1-4
Publish Date
1998
Start Page
103
End Page
111
DOI
10.1016/S1381-1177(98)00087-3

2-Keto-3-deoxy-6-phosphogalactonate Aldolase as a Catalyst for Stereocontrolled Carbon-Carbon Bond Formation

Authors
Henderson, DP; Cotterill, IC; Shelton, MC; Toone, EJ
MLA Citation
Henderson, DP, Cotterill, IC, Shelton, MC, and Toone, EJ. "2-Keto-3-deoxy-6-phosphogalactonate Aldolase as a Catalyst for Stereocontrolled Carbon-Carbon Bond Formation." Journal of Organic Chemistry 63.4 (1998): 906-907.
Source
scival
Published In
Journal of Organic Chemistry
Volume
63
Issue
4
Publish Date
1998
Start Page
906
End Page
907

Phosphate as Both a Activating and Protecting Group in Aldolase-Based Syntheses

Authors
Cotterill, IC; Shelton, MC; Henderson, DF; Toone, EJ
MLA Citation
Cotterill, IC, Shelton, MC, Henderson, DF, and Toone, EJ. "Phosphate as Both a Activating and Protecting Group in Aldolase-Based Syntheses." J. Chem. Soc. Perkin Trans. I 7 (1998): 1335-1342. (Academic Article)
Source
manual
Published In
J. Chem. Soc. Perkin Trans. I
Volume
7
Publish Date
1998
Start Page
1335
End Page
1342

Effect of phosphorylation on the reaction rate of unnatural electrophiles with 2-keto-3-deoxy-6-phosphogluconate aldolase

D-Glyceraldehyde is accepted as an electrophile by 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14) at 1% the rate of natural substrate, D-glyceraldehyde 3-phosphate. Accordingly, it was expected that addition of a phosphate moiety at C3 or C4 of unnatural aldehydes would enhance their activity as electrophilic substrates. Furthermore, phosphate would act as a useful protecting group during synthetic manipulations of the aldol adduct. A variety of phosphorylated and non-phosphorylated aldehydes were synthesized and evaluated as substrates for KDPG aldolase. Although small variations in reaction rate were observed, phosphorylation failed to provide a universal rate enhancement. Evaluation of substrate kinetic parameters revealed that the high rate of reaction of D-glyceraldehyde 3-phosphate compared to related electrophiles is entirely due to the efficiency of turnover with little change in binding exhibited among various substrates.

Authors
Cotterill, IC; Shelton, MC; Machemer, DEW; Henderson, DP; Toone, EJ
MLA Citation
Cotterill, IC, Shelton, MC, Machemer, DEW, Henderson, DP, and Toone, EJ. "Effect of phosphorylation on the reaction rate of unnatural electrophiles with 2-keto-3-deoxy-6-phosphogluconate aldolase." Journal of the Chemical Society - Perkin Transactions 1 7 (1998): 1335-1341.
Source
scival
Published In
Journal of the Chemical Society. Perkin transactions 1
Issue
7
Publish Date
1998
Start Page
1335
End Page
1341

Stereospecific Preparation of the N-Terminal Amino Acid Moiety of Nikkomycins K(X) and K(Z) via a Multiple Enzyme Synthesis.

Authors
Henderson, DP; Shelton, MC; Cotterill, IC; Toone, EJ
MLA Citation
Henderson, DP, Shelton, MC, Cotterill, IC, and Toone, EJ. "Stereospecific Preparation of the N-Terminal Amino Acid Moiety of Nikkomycins K(X) and K(Z) via a Multiple Enzyme Synthesis." J Org Chem 62.23 (November 14, 1997): 7910-7911.
PMID
11671888
Source
pubmed
Published In
The Journal of Organic Chemistry
Volume
62
Issue
23
Publish Date
1997
Start Page
7910
End Page
7911

(S)NO signals: translocation, regulation, and a consensus motif.

Authors
Stamler, JS; Toone, EJ; Lipton, SA; Sucher, NJ
MLA Citation
Stamler, JS, Toone, EJ, Lipton, SA, and Sucher, NJ. "(S)NO signals: translocation, regulation, and a consensus motif." Neuron 18.5 (May 1997): 691-696. (Review)
PMID
9182795
Source
pubmed
Published In
Neuron
Volume
18
Issue
5
Publish Date
1997
Start Page
691
End Page
696

Exploring the basis of peptide-carbohydrate crossreactivity: evidence for discrimination by peptides between closely related anti-carbohydrate antibodies.

To investigate the molecular basis of antigenic mimicry by peptides, we studied a panel of closely related mAbs directed against the cell-wall polysaccharide of group A Streptococcus. These antibodies have restricted V-gene usage, indicating a shared mechanism of binding to a single epitope. Epitope mapping studies using synthetic fragments of the cell-wall polysaccharide supported this conclusion. All of the mAbs isolated crossreactive peptides from a panel of phage-displayed libraries, and competition studies indicated that many of the peptides bind at or near the carbohydrate binding site. Surprisingly, the peptides isolated by each mAb fell into distinct consensus-sequence groups that discriminated between the mAbs, and in general, the peptides bound only to the mAbs used for their isolation. Similar results were obtained with polyclonal antibodies directed against synthetic oligosaccharide fragments of the streptococcal cell-wall polysaccharide. Thus, the peptides appear to be specific for their isolating antibodies and are not recognized by the same mechanism as their carbohydrate counterparts.

Authors
Harris, SL; Craig, L; Mehroke, JS; Rashed, M; Zwick, MB; Kenar, K; Toone, EJ; Greenspan, N; Auzanneau, FI; Marino-Albernas, JR; Pinto, BM; Scott, JK
MLA Citation
Harris, SL, Craig, L, Mehroke, JS, Rashed, M, Zwick, MB, Kenar, K, Toone, EJ, Greenspan, N, Auzanneau, FI, Marino-Albernas, JR, Pinto, BM, and Scott, JK. "Exploring the basis of peptide-carbohydrate crossreactivity: evidence for discrimination by peptides between closely related anti-carbohydrate antibodies." Proc Natl Acad Sci U S A 94.6 (March 18, 1997): 2454-2459.
PMID
9122216
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
94
Issue
6
Publish Date
1997
Start Page
2454
End Page
2459

Thermodynamic Solvent Isotope Effects and Molecular Hydorphobicity

Authors
Oas, TG; Toone, EJ
MLA Citation
Oas, TG, and Toone, EJ. "Thermodynamic Solvent Isotope Effects and Molecular Hydorphobicity." Adv. Biophys. Chem. 6 (1997): 1-52. (Academic Article)
Source
manual
Published In
Adv. Biophys. Chem.
Volume
6
Publish Date
1997
Start Page
1
End Page
52

(S)NO Signals: A Consensus Motif

Authors
Stamler, JS; Toone, EJ; Lipton, S; Sucher, N
MLA Citation
Stamler, JS, Toone, EJ, Lipton, S, and Sucher, N. "(S)NO Signals: A Consensus Motif." Neuron 18 (1997): 691-. (Academic Article)
Source
manual
Published In
Neuron
Volume
18
Publish Date
1997
Start Page
691

Synthesis of carbon-linked glycopeptides through catalytic asymmetric hydrogenation

Authors
Debenham, SD; Debenham, JS; Burk, MJ; Toone, EJ
MLA Citation
Debenham, SD, Debenham, JS, Burk, MJ, and Toone, EJ. "Synthesis of carbon-linked glycopeptides through catalytic asymmetric hydrogenation." Journal of the American Chemical Society 119.41 (1997): 9897-9898.
Source
scival
Published In
Journal of the American Chemical Society
Volume
119
Issue
41
Publish Date
1997
Start Page
9897
End Page
9898
DOI
10.1021/ja971324z

A comparison of the fine saccharide-binding specificity of Dioclea grandiflora lectin and concanavalin A.

The lectin from the seeds of Dioclea grandiflora (DGL) is a Man/Glc-specific tetrameric protein with physical and saccharide-binding properties reported to be similar to that of the jack bean lectin concanavalin A (ConA). Unlike other plant lectins, both DGL and ConA bind with high affinity to the core trimannoside moiety, 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, which is present in all asparagine-linked carbohydrates. In the present study, hemagglutination inhibition techniques have been used to investigate binding of DGL and ConA to a series of mono- and dideoxy analogs of methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside and to a series of asparagine-linked oligomannose and complex oligosaccharides and glycopeptides. The results indicate that both DGL and ConA recognize epitopes on all three residues of the trimannoside: the 3-, 4-, and 6-hydroxyl groups of the alpha(1-6)Man residue, the 3-hydroxyl group of the alpha(1-3)Man residue, and the 2- and 4-hydroxyl groups of the central Man residue of the core trimannoside. However, unlike ConA, DGL does not bind to biantennary complex carbohydrates. This was confirmed by showing that biantennary complex glycopeptides do not bind to a DGL-Sepharose affinity column. Unlike ConA, DGL does not show enhanced affinity for a large N-linked oligomannose carbohydrate (Man9 glycopeptide) relative to the trimannoside. Thus, DGL and ConA share similar epitope recognition of the core trimannoside moiety. However, they exhibit differences in their fine specificities for larger N-linked oligomannose and complex carbohydrates.

Authors
Gupta, D; Oscarson, S; Raju, TS; Stanley, P; Toone, EJ; Brewer, CF
MLA Citation
Gupta, D, Oscarson, S, Raju, TS, Stanley, P, Toone, EJ, and Brewer, CF. "A comparison of the fine saccharide-binding specificity of Dioclea grandiflora lectin and concanavalin A." Eur J Biochem 242.2 (December 1, 1996): 320-326.
PMID
8973650
Source
pubmed
Published In
European journal of biochemistry / FEBS
Volume
242
Issue
2
Publish Date
1996
Start Page
320
End Page
326

Analysis of the binding specificities of oligomannoside-binding proteins using methylated monosaccharides.

The binding specificities of the closely related lectins from Canavalia ensiformis and Dioclea grandiflora were examined using specifically O-alkylated mono- and disaccharides. Both lectins accept any substitution at the monosaccharide C2 hydroxyl group. The binding energy of C2-alkylated ligands-concanavalin A complexes increases by 1 kcal mol-1 for the C2-O-ethyl ligand, while the binding energies of the corresponding complexes with the Dioclea lectin are identical. Both lectins accept methyl, but not ethyl, substitution of the C3 hydroxyl, in contrast to earlier reports. The results are interpreted in terms of existing models of the concanavalin A binding site. While the results are consistent with a model of the concanavalin A extended binding site that places the non-reducing terminus of all disaccharides in the monosaccharide binding site, they point to the dangers of interpreting the binding behavior of unnatural saccharide ligands on the basis of crystallographic data obtained with native ligands.

Authors
Chervenak, MC; Toone, EJ
MLA Citation
Chervenak, MC, and Toone, EJ. "Analysis of the binding specificities of oligomannoside-binding proteins using methylated monosaccharides." Bioorg Med Chem 4.11 (November 1996): 1963-1977.
PMID
9007280
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry
Volume
4
Issue
11
Publish Date
1996
Start Page
1963
End Page
1977

Specificity of C-glycoside complexation by mannose/glucose specific lectins.

The binding of the mannose/glucose specific lectins from Canavalia ensiformis (concanavalin A) and Dioclea grandiflora to a series of C-glucosides were studied by titration microcalorimetry and fluorescence anisotropy titration. These closely related lectins share a specificity for the trimannoside methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, and are a useful model system for addressing the feasibility of differentiating between lectins with overlapping carbohydrate specificities. The ligands were designed to address two issues: (1) how the recognition properties of non-hydrolyzable C-glycoside analogues compare with those of the corresponding O-glycosides and (2) the effect of presentation of more than one saccharide recognition epitope on both affinity and specificity. Both lectins bind the C-glycosides with affinities comparable to those of the O-glycoside analogues; however, the ability of both lectins to differentiate between gluco and manno diastereomers was diminished in the C-glycoside series. Bivalent norbornyl C-glycoside esters were bound by the lectin from Canavalia but only weakly by the lectin from Dioclea. In addition to binding the bivalent ligands, concanavalin A discriminated between C-2 epimers, with the manno configuration binding more tightly than the gluco. The stoichiometry of binding of the bivalent ligands to both di- and tetrameric lectin was two binding sites per ligand, rather than the expected 1:1 stoichiometry. Together, these results suggest that concanavalin A may possess more than one class of carbohydrate binding sites and that these additional sites show stereochemical discrimination similar to that of the previously identified monosaccharide binding site. The implications of these findings for possible in vivo roles of plant lectins and for the use of concanavalin A as a research tool are discussed.

Authors
Weatherman, RV; Mortell, KH; Chervenak, M; Kiessling, LL; Toone, EJ
MLA Citation
Weatherman, RV, Mortell, KH, Chervenak, M, Kiessling, LL, and Toone, EJ. "Specificity of C-glycoside complexation by mannose/glucose specific lectins." Biochemistry 35.11 (March 19, 1996): 3619-3624.
PMID
8639514
Source
pubmed
Published In
Biochemistry
Volume
35
Issue
11
Publish Date
1996
Start Page
3619
End Page
3624
DOI
10.1021/bi951916z

Phenylalanine 30 plays an important role in receptor binding of verotoxin-1.

The homopentameric B subunit of verotoxin 1 (VT1) binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). We produced mutants with alanine substitutions for residues found near the cleft between adjacent subunits. Substitution of alanine for phenylalanine 30 (Phe-30) resulted in a fourfold reduction in B subunit binding affinity for Gb3 and a 10-fold reduction in receptor density in a solid-phase binding assay. The interaction of wild-type and mutant B subunits with Pk trisaccharide in solution was examined by titration microcalorimetry. The carbohydrate binding of the mutant was markedly impaired compared with that of the wild type and was too weak to allow calculation of a binding constant. These results demonstrate that the mutation significantly impaired the carbohydrate-binding function of the B subunit. To ensure that the mutation had not caused a significant change in structure, the mutant B subunit was crystallized and its structure was determined by X-ray diffraction. Difference Fourier analysis showed that its structure was identical to that of the wild type, except for the substitution of alanine for Phe-30. The mutation was also produced in the VT1 operon, and mutant holotoxin was purified to homogeneity. The cytotoxicity of the mutant holotoxin was reduced by a factor of 10(5) compared to that of the wild type in the Vero cell cytotoxicity assay. The results suggest that the aromatic ring of Phe-30 plays a major role in binding of the B subunit to the Galalpha1-4Galbeta1-4Glc trisaccharide portion of Gb3. Examination of the VT1 B crystal structure suggests two potential carbohydrate-binding sites which lie on either side of Phe-30.

Authors
Clark, C; Bast, D; Sharp, AM; St Hilaire, PM; Agha, R; Stein, PE; Toone, EJ; Read, RJ; Brunton, JL
MLA Citation
Clark, C, Bast, D, Sharp, AM, St Hilaire, PM, Agha, R, Stein, PE, Toone, EJ, Read, RJ, and Brunton, JL. "Phenylalanine 30 plays an important role in receptor binding of verotoxin-1." Mol Microbiol 19.4 (February 1996): 891-899.
PMID
8820657
Source
pubmed
Published In
Molecular Microbiology
Volume
19
Issue
4
Publish Date
1996
Start Page
891
End Page
899

Keto-3-Deoxy-6-Phosphogluconate Aldolases as Catalysts for Stereocontrolled Carbon-Carbon Bond Formation

Authors
Shelton, MC; Cotterill, IC; Novak, STA; Poonawala, RM; Sudarshan, S; Toone, EJ
MLA Citation
Shelton, MC, Cotterill, IC, Novak, STA, Poonawala, RM, Sudarshan, S, and Toone, EJ. "Keto-3-Deoxy-6-Phosphogluconate Aldolases as Catalysts for Stereocontrolled Carbon-Carbon Bond Formation." J. Am. Chem. Soc. 118 (1996): 2117-2125. (Academic Article)
Source
manual
Published In
J. Am. Chem. Soc.
Volume
118
Publish Date
1996
Start Page
2117
End Page
2125

2-keto-3-deoxy-6-phosphogluconate aldolases as catalysts for stereocontrolled carbon-carbon bond formation

The pyruvate aldolases use pyruvate as the nucleophilic component in stereoselective aldol condensations, producing a 4-hydroxy-2-ketobutyrate framework. We have examined the 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Pseudomonas putida, Escherichia coli, and Zymomonas mobilis for utility as synthetic reagents. Unlike other pyruvate aldolases examined to date, the KDPG aldolases accept short-chain, non-carbohydrate electrophilic aldehydes as substrates, providing a general methodology for the construction of the 4-hydroxy-2-ketobutyrate skeleton. The three aldolases differ markedly with respect to enzyme stability, pH optima, stability in organic cosolvent mixtures, substrate specificity, and diastereoselectivity during aldol condensation. All three enzymes show broad substrate specificity with regard to the electrophilic component. The primary requirements for substrate activity appear to be minimal steric hindrance and the presence of electron-withdrawing substituents at C2. The aldolases from Pseudomonas and Escherichia are also specific for the D-stereochemical configuration at C2, while the enzyme from Zymomonas displays no stereochemical discrimination with regard to the electrophilic substrate. Nucleophiles other than pyruvate are accepted as nucleophilic substrates by all three enzymes, provided the electrophile is sufficiently reactive. In preparative scale reactions with three unnatural electrophiles, the three enzymes show varying degrees of stereochemical fidelity. In most cases, a single diastereomer of the aldol adduct was produced, although in one case, a diastereomeric excess of 50% was observed. In all cases, the diastereoselectivity is exclusively kinetic in origin, despite the reversibility of some reactions. The enzymes are remarkably tolerant of added cosolvent: all three showed >60% of native activity in 30% DMSO and DMF. By appropriate choice of enzyme, the KDPG aldolases offer exceptional utility for stereocontrolled carbon-carbon bond formation under a wide range of experimental conditions.

Authors
Shelton, MC; Cotterill, IC; Novak, STA; Poonawala, RM; Sudarshan, S; Toone, EJ
MLA Citation
Shelton, MC, Cotterill, IC, Novak, STA, Poonawala, RM, Sudarshan, S, and Toone, EJ. "2-keto-3-deoxy-6-phosphogluconate aldolases as catalysts for stereocontrolled carbon-carbon bond formation." Journal of the American Chemical Society 118.9 (1996): X-2125.
Source
scival
Published In
Journal of the American Chemical Society
Volume
118
Issue
9
Publish Date
1996
Start Page
X
End Page
2125

2-Keto-3-deoxy-6-phosphogluconate aldolases as catalysts for stereocontrolled carbon-carbon bond formation

The pyruvate aldolases use pyruvate as the nucleophilic component in stereoselective aldol condensations, producing a 4-hydroxy-2-ketobutyrate framework. We have examined the 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Pseudomonas putida, Escherichia coli, and Zymomonas mobilis for utility as synthetic reagents. Unlike other pyruvate aldolases examined to date, the KDPG aldolases accept short-chain, non-carbohydrate electrophilic aldehydes as substrates, providing a general methodology for the construction of the 4-hydroxy-2-ketobutyrate skeleton. The three aldolases differ markedly with respect to enzyme stability, pH optima, stability in organic cosolvent mixtures, substrate specificity, and diastereoselectivity during aldol condensation. All three enzymes show broad substrate specificity with regard to the electrophilic component. The primary requirements for substrate activity appear to be minimal steric hindrance and the presence of electron-withdrawing substituents at C2. The aldolases from Pseudomonas and Escherichia are also specific for the D-stereochemical configuration at C2, while the enzyme from Zymomonas displays no stereochemical discrimination with regard to the electrophilic substrate. Nucleophiles other than pyruvate are accepted as nucleophilic substrates by all three enzymes, provided the electrophile is sufficiently reactive. In preparative scale reactions with three unnatural electrophiles, the three enzymes show varying degrees of stereochemical fidelity. In most cases, a single diastereomer of the aldol adduct was produced, although in one case, a diastereomeric excess of 50% was observed. In all cases, the diastereoselectivity is exclusively kinetic in origin, despite the reversibility of some reactions. The enzymes are remarkably tolerant of added cosolvent: all three showed >60% of native activity in 30% DMSO and DMF. By appropriate choice of enzyme, the KDPG aldolases offer exceptional utility for stereocontrolled carbon-carbon bond formation under a wide range of experimental conditions.

Authors
Shelton, MC; Cotterill, IC; Novak, STA; Poonawala, RM; Sudarshan, S; Toone, EJ
MLA Citation
Shelton, MC, Cotterill, IC, Novak, STA, Poonawala, RM, Sudarshan, S, and Toone, EJ. "2-Keto-3-deoxy-6-phosphogluconate aldolases as catalysts for stereocontrolled carbon-carbon bond formation." Journal of the American Chemical Society 118.9 (1996): 2117-2125.
Source
scival
Published In
Journal of the American Chemical Society
Volume
118
Issue
9
Publish Date
1996
Start Page
2117
End Page
2125
DOI
10.1021/ja952596+

Calorimetric analysis of the binding of lectins with overlapping carbohydrate-binding ligand specificities.

The thermodynamics of binding of a system of plant lectins specific for the oligosaccharide methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside have been studied calorimetrically. This system of lectins consists of concanavalin A, the lectin isolated from Dioclea grandiflora, and the lectin from Galanthus nivalis. The group thus contains lectins with similar structures and similar binding properties as well as lectins with different structures but similar binding properties. Concanavalin A and the lectin from Dioclea are highly homologous, while the lectin from Galanthus nivalis shares no sequence homology with either of the legume lectins, although it also binds the mannose trisaccharide tightly. Calorimetric data for oligosaccharide binding to both of the legume lectins suggests that the total binding site comprises a single high-affinity site and an additional extended site. The pattern of binding for the lectin from Galanthus is significantly different. Binding studies with the same saccharides indicate that the lectin has binding sites designed specifically for the 1-->3 and 1-->6 arms of the mannose trisaccharide that are unable to accommodate other saccharides. Enthalpy--entropy compensation was observed for several saccharides as a function of lectin structure. Contributions of solvation effects to the enthalpy of binding and the configurational entropies were determined experimentally. For those systems studied here, solute-solute attractive interactions and configurational entropies were the greatest contributors to enthalpy-entropy compensation. Our studies clearly demonstrate that, despite their common affinity for the mannose trisaccharide, the three lectins bind oligosaccharides very differently.

Authors
Chervenak, MC; Toone, EJ
MLA Citation
Chervenak, MC, and Toone, EJ. "Calorimetric analysis of the binding of lectins with overlapping carbohydrate-binding ligand specificities." Biochemistry 34.16 (April 25, 1995): 5685-5695.
PMID
7727428
Source
pubmed
Published In
Biochemistry
Volume
34
Issue
16
Publish Date
1995
Start Page
5685
End Page
5695

Differential dye-ligand chromatography as a general purification protocol for 2-keto-3-deoxy-6-phosphogluconate aldolases

2-Keto-3-deoxy-6-phosphogluconate aldolases (KDPG aldolases, EC 4.1.2.14) from four sources, Esherichia coli, Pseudomonas putida, Pseudomonas saccharophila, and Zymononas mobilis, were purified from 17- to 48- fold by differential dye-ligand chromatography (DD-L chromatography). This technique, using Procion Navy H-ER (150 grain) as a negative column and Procion Yellow MX-GR as a positive column supported on Sepharose CL-4B, represents a general methodology for the rapid, facile purification of KDPG aldolases from disparate microbial sources. © 1994.

Authors
Shelton, MC; Toone, EJ
MLA Citation
Shelton, MC, and Toone, EJ. "Differential dye-ligand chromatography as a general purification protocol for 2-keto-3-deoxy-6-phosphogluconate aldolases." Tetrahedron: Asymmetry 6.1 (1995): 207-211.
Source
scival
Published In
Tetrahedron: Asymmetry
Volume
6
Issue
1
Publish Date
1995
Start Page
207
End Page
211

Spatial organization versus total surface area as a predictor of protein hydrophobicity. The hydrophobicity of the concanavalin A binding site

Authors
Isbister, BD; Hilaire, PMS; Toone, EJ
MLA Citation
Isbister, BD, Hilaire, PMS, and Toone, EJ. "Spatial organization versus total surface area as a predictor of protein hydrophobicity. The hydrophobicity of the concanavalin A binding site." Journal of the American Chemical Society 117.51 (1995): 12877-12878.
Source
scival
Published In
Journal of the American Chemical Society
Volume
117
Issue
51
Publish Date
1995
Start Page
12877
End Page
12878
DOI
10.1021/ja00156a033

Erratum: Calorimetric analysis of the binding of lectins with overlapping carbohydrate-binding ligand specificities (Biochemistry (April 25, 1995) 34:16 (5685-5695))

Authors
Chervenak, MC; Toone, EJ
MLA Citation
Chervenak, MC, and Toone, EJ. "Erratum: Calorimetric analysis of the binding of lectins with overlapping carbohydrate-binding ligand specificities (Biochemistry (April 25, 1995) 34:16 (5685-5695))." Biochemistry 34.24 (1995): 7966--.
Source
scival
Published In
Biochemistry
Volume
34
Issue
24
Publish Date
1995
Start Page
7966-
DOI
10.1021/bi00024a022

Interaction of the Shiga-like toxin type 1 B-subunit with its carbohydrate receptor.

A study of the binding of the Shiga-like toxin 1 (SLT-1) to the P(k) trisaccharide [methyl 4-O-(4-O-alpha-D-galactopyranosyl)-4-O-beta-D- glucopyranoside] and its constituent dissacharides was carried out. The trisaccharide represents the carbohydrate recognition domain of the neutral glycolipid receptor of the SLT-1, globotriosylceramide (GbOse3). The binding constant for soluble trisaccharide to the soluble pentameric B-subunit is weak, with a K(a) of (0.5-1) x 10(3) M-1 for B-subunit monomer. Scatchard analysis of the binding data indicates five identical non-interacting carbohydrate binding sites per B-subunit pentamer and no cooperativity in binding. Despite weak binding (delta G = -3.6 kcal mol-1), the enthalpy of binding (delta H = -12 kcal mol-1) and the change in molar heat capacity accompanying binding (delta C(p) = -40 eu) are comparable to other protein-carbohydrate interactions. Dynamic light scattering studies indicate that carbohydrate binding induces protein aggregation. At carbohydrate concentrations where > 90% of B-subunit monomers are bound, the far-UV CD spectra were unchanged, whereas a change in the near-UV CD, maximal near 270 nm, titrated to give an apparent binding constant in good agreement with that obtained by isothermal microcalorimetry. Steady-state fluorescence and fluorescence lifetime measurements indicated that the environments of the central tryptophans are perturbed during saccharide binding, and the changes correlate with the extent of protein aggregation. On the basis of the thermodynamics of binding, optical spectroscopy, and binding-induced aggregation, we propose a model of SLT-1-membrane interaction that relies on protein-carbohydrate interaction for specificity and protein-lipid interaction for tight binding.

Authors
St Hilaire, PM; Boyd, MK; Toone, EJ
MLA Citation
St Hilaire, PM, Boyd, MK, and Toone, EJ. "Interaction of the Shiga-like toxin type 1 B-subunit with its carbohydrate receptor." Biochemistry 33.48 (December 6, 1994): 14452-14463.
PMID
7981205
Source
pubmed
Published In
Biochemistry
Volume
33
Issue
48
Publish Date
1994
Start Page
14452
End Page
14463

Increased adhesion between neutral lipid bilayers: interbilayer bridges formed by tannic acid.

Tannic acid (TA) is a naturally occurring polyphenolic compound that aggregates membranes and neutral phosolipid vesicles and precipitates many proteins. This study analyzes TA binding to lipid membranes and the ensuing aggregation. The optical density of dispersions of phosphatidylcholine (PC) vesicles increased upon the addition of TA and electron micrographs showed that TA caused the vesicles to aggregate and form stacks of tightly packed disks. Solution calorimetry showed that TA bound to PC bilayers with a molar binding enthalpy of -8.3 kcal/mol and zeta potential measurements revealed that TA imparted a small negative charge to PC vesicles. Monolayer studies showed that TA bound to PC with a dissociation constant of 1.5 microM and reduced the dipole potential by up to 250 mV. Both the increase in optical density and decrease in dipole potential produced by TA could be reversed by the addition of polyvinylpyrrolidone, a compound that chelates TA by providing H-bond acceptor groups. NMR, micropipette aspiration, and x-ray diffraction experiments showed that TA incorporated into liquid crystalline PC membranes, increasing the area per lipid molecule and decreasing the bilayer thickness by 2 to 4%. 2H-NMR quadrupole splitting measurements also showed that TA associated with a PC molecule for times much less than 10(-4) s. In gel phase bilayers, TA caused the hydrocarbon chains from apposing monolayers to fully interdigitate. X-ray diffraction measurements of both gel and liquid crystalline dispersions showed that TA, at a critical concentration of about 1 mM, reduced the fluid spacing between adjacent bilayers by 8-10 A. These data place severe constraints on how TA can pack between adjacent bilayers and cause vesicles to adhere. We conclude that TA promotes vesicle aggregation by reducing the fluid spacing between bilayers by the formation of transient interbilayer bridges by inserting its digallic acid residues into the interfacial regions of adjacent bilayers and spanning the interbilayer space.

Authors
Simon, SA; Disalvo, EA; Gawrisch, K; Borovyagin, V; Toone, E; Schiffman, SS; Needham, D; McIntosh, TJ
MLA Citation
Simon, SA, Disalvo, EA, Gawrisch, K, Borovyagin, V, Toone, E, Schiffman, SS, Needham, D, and McIntosh, TJ. "Increased adhesion between neutral lipid bilayers: interbilayer bridges formed by tannic acid." Biophys J 66.6 (June 1994): 1943-1958.
PMID
8075329
Source
pubmed
Published In
Biophysical Journal
Volume
66
Issue
6
Publish Date
1994
Start Page
1943
End Page
1958
DOI
10.1016/S0006-3495(94)80988-9

Preface

Authors
Dodds, DR; Toone, PEJ
MLA Citation
Dodds, DR, and Toone, PEJ. "Preface." January 1, 1994. xiii-.
Source
scopus
Volume
2
Publish Date
1994
Start Page
xiii
DOI
10.1016/0968-0896(94)80002-2

A Molecular Mechanism for Increasing Adhesion Between Neutral Lipid Membranes: Interbilayer Bridges Formed by Tannic Acid

Authors
Simon, SA; Sisalvo, EA; Gawrisch, K; Borovyagin, V; Toone, EJ; Schiffman, SS; Needham, D; McIntosh, TJ
MLA Citation
Simon, SA, Sisalvo, EA, Gawrisch, K, Borovyagin, V, Toone, EJ, Schiffman, SS, Needham, D, and McIntosh, TJ. "A Molecular Mechanism for Increasing Adhesion Between Neutral Lipid Membranes: Interbilayer Bridges Formed by Tannic Acid." Biophys. J. 66 (1994): 1943-1957. (Academic Article)
Source
manual
Published In
Biophys. J.
Volume
66
Publish Date
1994
Start Page
1943
End Page
1957

A direct measure of the contribution of solvent reorganization to the enthalpy of ligand binding

The thermodynamics of association of several binding systems, including protein-carbohydrate, small molecule-small molecule, protein-peptide, and protein-nucleic acid, were evaluated calorimetrically in light and heavy water. In every case, the enthalpy of binding in D2O was decreased relative to that in H2O: the differences range from 400 to 1800 cal mol-1. A compensating change in AS left the free energy of binding virtually unchanged in each case. A strong correlation between the differential enthalpy of binding and ΔCP for binding was observed, with a slope of 5 K. An analysis of the observed effect utilizing a Born-Haber thermodynamic cycle shows that the measured decrease in enthalpy represents approximately 10% of the binding enthalpy arising from solvent reorganization. For the range of systems investigated, solvent reorganization provides 25-100% of the observed enthalpy of binding.

Authors
Chervenak, MC; Toone, EJ
MLA Citation
Chervenak, MC, and Toone, EJ. "A direct measure of the contribution of solvent reorganization to the enthalpy of ligand binding." Journal of the American Chemical Society 116.23 (1994): 10533-10539.
Source
scival
Published In
Journal of the American Chemical Society
Volume
116
Issue
23
Publish Date
1994
Start Page
10533
End Page
10539

Structure and energetics of protein-carbohydrate complexes

During the past year, several new coordinate sets for protein-carbohydrate complexes have appeared. The data fall into two important categories: complexes of lectins other than legume and chemotaxis proteins, and high-resolution refinements that allow placement of discrete water molecules. These data should lead to a better understanding of the role of water in protein-carbohydrate complexation. Interest in the energetics of association is growing, and calorimetrically determined thermodynamic parameters are now available for a host of protein-carbohydrate complexations. The impact of recent structural and energetic studies on proposed models of protein-carbohydrate association is discussed.

Authors
Toone, EJ
MLA Citation
Toone, EJ. "Structure and energetics of protein-carbohydrate complexes." Current Opinion in Structural Biology 4.5 (1994): 719-728.
Source
scival
Published In
Current Opinion in Structural Biology
Volume
4
Issue
5
Publish Date
1994
Start Page
719
End Page
728
DOI
10.1016/S0959-440X(94)90170-8

Preface

Authors
Dodds, DR; Toone, PEJ
MLA Citation
Dodds, DR, and Toone, PEJ. "Preface." Bioorganic and Medicinal Chemistry 2.6 (1994): xiii-.
Source
scival
Published In
Bioorganic & Medicinal Chemistry
Volume
2
Issue
6
Publish Date
1994
Start Page
xiii

Calorimetric evaluation of enzyme kinetic parameters

The measurement of kinetic parameters (kcat, Km, Ki) for a wide range of proteolytic enzymes is vital to contemporary bioorganic and medicinal chemistry. Enzyme assays based on changes in optical properties of the system or changes in concentration of an ion detectable electrochemically are not viable for many enzyme-catalyzed reactions, including proteases and peptidases. Hydrolysis of an amide bond produces no change in the optical properties or pH of the reaction solution, and as a result no general direct method for the evaluation of protease kinetics exists using underivatized substrates. We report here a microcalorimetric assay which provides a general and straightforward technique for the measurement of kinetic parameters of hydrolysis of underivatized peptide substrates by proteases. Using this technique, kcat values as high as 10-s-1 can be easily measured. We demonstrate the utility of the technique by measuring the kinetics of hydrolysis of several N-acylamino acids by the synthetically useful enzyme hog kidney acylase and the hydrolysis of tetrapeptide p-nitrophenyl anilides by subtilisin BPN′. Although we have used the technique to monitor amide bond hydrolysis, the methodology is applicable to any system with appropriate kinetic and thermodynamic properties. © 1993 American Chemical Society.

Authors
Williams, BA; Toone, EJ
MLA Citation
Williams, BA, and Toone, EJ. "Calorimetric evaluation of enzyme kinetic parameters." Journal of Organic Chemistry 58.13 (1993): 3507-3510.
Source
scival
Published In
The Journal of Organic Chemistry
Volume
58
Issue
13
Publish Date
1993
Start Page
3507
End Page
3510

Energetics of lectin-carbohydrate binding. A microcalorimetric investigation of concanavalin A-oligomannoside complexation.

Despite years of study, a comprehensive picture of the binding of the lectin from Canavalia ensiformis, concanavalin A, to carbohydrates remains elusive. We report here studies on the interaction of concanavalin A with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, the minimum carbohydrate epitope that completely fills the oligosaccharide binding site, and the two conceptual disaccharide "halves" of the trisaccharide, methyl 3-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside and methyl 6-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, using titration microcalorimetry. In all cases the interaction of protein and carbohydrate is enthalpically driven, with an unfavorable entropic contribution. The choice of concentration scales has an important impact on both the magnitude and, in some cases, the sign of the entropic component of the free energy of binding. The thermodynamic data suggest binding of the two disaccharides may take place in distinct sites, as opposed to binding in a single high affinity site. In contrast to carbohydrate-antibody binding, delta Cp values were small and negative, pointing to possible differences in the motifs used by the two groups of proteins to bind carbohydrates. The thermodynamic data are interpreted in terms of solvent reorganization. Cooperativity during lectin-carbohydrate binding was also investigated. Significant cooperativity was observed only for binding of the trisaccharide, and gave a Hill plot coefficient of 1.3 for dimeric protein.

Authors
Williams, BA; Chervenak, MC; Toone, EJ
MLA Citation
Williams, BA, Chervenak, MC, and Toone, EJ. "Energetics of lectin-carbohydrate binding. A microcalorimetric investigation of concanavalin A-oligomannoside complexation." J Biol Chem 267.32 (November 15, 1992): 22907-22911.
PMID
1429640
Source
pubmed
Published In
The Journal of biological chemistry
Volume
267
Issue
32
Publish Date
1992
Start Page
22907
End Page
22911

Pyruvate aldolases as reagents for stereospecific aldol condensation

KDPG aldolase, a representative member of the largest but as of yet unexplored group of aldolases which utilize pyruvate as the nucleophilic component in aldol condensation, accepts a number of unnatural aldehydes as electrophiles in stereospecific aldol condensation, providing access to highly and differentially functionalized α-keto acid products. © 1992 American Chemical Society.

Authors
Allen, ST; Heintzelman, GR; Toone, EJ
MLA Citation
Allen, ST, Heintzelman, GR, and Toone, EJ. "Pyruvate aldolases as reagents for stereospecific aldol condensation." Journal of Organic Chemistry 57.2 (1992): 426-427.
Source
scival
Published In
The Journal of Organic Chemistry
Volume
57
Issue
2
Publish Date
1992
Start Page
426
End Page
427

Enzymes in organic synthesis 49. Resolutions of racemic monocyclic esters with pig liver esterase.

Pig liver esterase (PLE)- catalyzed hydrolyses of the racemic methyl esters of cyclobutane-, cyclohexane-, and cyclohex-4-ene-carboxylic acids bearing cis-2-methyl or cis-2-bromomethyl substituents are highly stereoselective, giving the corresponding acid products of ≥ 97% ee. The stereoselectivity of the enzyme exhibits the expected reversal for such compounds, with the absolute configurations of the cyclobutane and cyclohexane acids being of the opposite absolute configuration types, and cyclopentane substrates such as cis-1-carbomethoxy-2-methylcyclopentane representing the change-over structures and giving products of only 22%ee. This stereoselectivity reversal, and the absolute configurations preferred, are as predicted by the recently proposed active site model for the enzyme. © 1993.

Authors
Toone, EJ; Jones, JB
MLA Citation
Toone, EJ, and Jones, JB. "Enzymes in organic synthesis 49. Resolutions of racemic monocyclic esters with pig liver esterase." Tetrahedron: Asymmetry 2.3 (1991): 207-222.
Source
scival
Published In
Tetrahedron: Asymmetry
Volume
2
Issue
3
Publish Date
1991
Start Page
207
End Page
222

Enzymes in Organic Synthesis. 50. Probing the Dimensions of the Large Hydrophobic Binding Region of the Active Site of Pig Liver Esterase Using Substituted Aryl Malonate Substrates

Authors
Toone, EJ; Jones, JB
MLA Citation
Toone, EJ, and Jones, JB. "Enzymes in Organic Synthesis. 50. Probing the Dimensions of the Large Hydrophobic Binding Region of the Active Site of Pig Liver Esterase Using Substituted Aryl Malonate Substrates." Tetrahedron Asym. 2 (1991): 207-. (Academic Article)
Source
manual
Published In
Tetrahedron Asym.
Volume
2
Publish Date
1991
Start Page
207

Enzymatic synthesis of uridine 5′-diphosphoglucuronic acid on a gram scale

A practical route to uridine 5′-diphosphoglucuronic acid (UDP-GlcUA) from uridine 5′-diphosphoglucose (UDP-Glc) on a 1-g scale has been developed using uridine 5′-diphosphoglucose dehydrogenase (UDP-Glc DH, EC 1.1.1.22) from bovine liver. Crude UDP-Glc dehydrogenase was isolated from beef liver (450 units from 2.4 kg of frozen liver). Commercially available UDP-Glc dehydrogenase as well as a preparation from calf liver acetone powder were also evaluated as catalysts for large-scale production of UDP-GlcUA: both preparations exhibited too little activity to be synthetically useful. A platinum-catalyzed oxygen oxidation of UDP-Glc was also examined as a possible route to UDP-GlcUA: enzymatic oxidation was superior. These results establish a route to another of the important activated monosaccharides required for cell-free enzymatic syntheses of mammalian oligo- and polysaccharides. © 1991 American Chemical Society.

Authors
Toone, EJ; Simon, ES; Whitesides, GM
MLA Citation
Toone, EJ, Simon, ES, and Whitesides, GM. "Enzymatic synthesis of uridine 5′-diphosphoglucuronic acid on a gram scale." Journal of Organic Chemistry 56.19 (1991): 5603-5606.
Source
scival
Published In
The Journal of Organic Chemistry
Volume
56
Issue
19
Publish Date
1991
Start Page
5603
End Page
5606

Enzymes in organic synthesis 50. Probing the dimensions of the large hydrophobic binding region of the active site of pig liver esterase using substituted aryl malonate substrates

The active site model reported recently for the synthetically useful enzyme pig liver esterase (PILE) permits the structural specificity and stereoselectivity of the enzyme to be interpreted and predicted for a wide range of substrates. The specifications of the dimensions of this model were based on the specificity data available at that time. In order to test the model further, and to delineate more accurately the dimensions of its large hydrophobic (HL) binding pocket, PLE-catalyzed hydrolyses of dimethyl ortho- and para-methyl-, ethyl-, isopropyl-, and tert-butylphenyl 2-methylmalonate substrates have been carried out. Each of these malonate diesters proved to be a good substrate of the enzyme. In every case, the pro-S ester group was hydrolyzed to give R-acid-ester products of 78->-97% ee. The results show that the initial volume specified for the HL pocket was too small to accommodate the larger aryl groups of this substrate series. A modified model with an appropriately enlarged HL region is presented. The sizes of the other binding pockets remain unchanged. © 1991.

Authors
Toone, EJ; Jones, JB
MLA Citation
Toone, EJ, and Jones, JB. "Enzymes in organic synthesis 50. Probing the dimensions of the large hydrophobic binding region of the active site of pig liver esterase using substituted aryl malonate substrates." Tetrahedron: Asymmetry 2.10 (1991): 1041-1052.
Source
scival
Published In
Tetrahedron: Asymmetry
Volume
2
Issue
10
Publish Date
1991
Start Page
1041
End Page
1052

ENZYMES IN ORGANIC-SYNTHESIS .47. ACTIVE-SITE MODEL FOR INTERPRETING AND PREDICTING THE SPECIFICITY OF PIG-LIVER ESTERASE

Authors
TOONE, EJ; WERTH, MJ; JONES, JB
MLA Citation
TOONE, EJ, WERTH, MJ, and JONES, JB. "ENZYMES IN ORGANIC-SYNTHESIS .47. ACTIVE-SITE MODEL FOR INTERPRETING AND PREDICTING THE SPECIFICITY OF PIG-LIVER ESTERASE." JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 112.12 (June 6, 1990): 4946-4952.
Source
wos-lite
Published In
Journal of the American Chemical Society
Volume
112
Issue
12
Publish Date
1990
Start Page
4946
End Page
4952
DOI
10.1021/ja00168a047

Overproduction and substrate specificity of a bacterial fuculose-1-phosphate aldolase: A new enzymatic catalyst for stereocontrolled aldol condensation

Authors
Ozaki, A; Toone, EJ; Osten, CHVD; Sinskey, AJ; Whitesides, GM
MLA Citation
Ozaki, A, Toone, EJ, Osten, CHVD, Sinskey, AJ, and Whitesides, GM. "Overproduction and substrate specificity of a bacterial fuculose-1-phosphate aldolase: A new enzymatic catalyst for stereocontrolled aldol condensation." Journal of the American Chemical Society 112.12 (1990): 4970-4971.
Source
scival
Published In
Journal of the American Chemical Society
Volume
112
Issue
12
Publish Date
1990
Start Page
4970
End Page
4971

Molecular cloning of aldolases for synthetic applications

Authors
Osten, CHVD; Gioannetti, C; Barbas, C; Pederson, RL; Wang, Y-F; Wong, C-H; Ozaki, A; Toone, E; Whitesides, GM; Sinskey, AJ
MLA Citation
Osten, CHVD, Gioannetti, C, Barbas, C, Pederson, RL, Wang, Y-F, Wong, C-H, Ozaki, A, Toone, E, Whitesides, GM, and Sinskey, AJ. "Molecular cloning of aldolases for synthetic applications." Annals of the New York Academy of Sciences 613 (1990): 771-775.
PMID
2076016
Source
scival
Published In
Annals of the New York Academy of Sciences
Volume
613
Publish Date
1990
Start Page
771
End Page
775

Active-site model for interpreting and predicting the specificity of pig liver esterase

Pig liver esterase (PLE) is one of the useful enzymes for the preparation of valuable chiral synthons. However, its applications in asymmetric synthesis have been hampered by its seemingly unpredictable specificity. This disadvantage has now been overcome through the development of a simple and easy-to-use active-site model. The model, which is based on cubic-space descriptors, accounts for the structural selectivity and stereoselectivity of PLE-catalyzed hydrolyses reported so far and is also of predictive value for new substrate structures. The development and specification of the model, and its application in analyzing the specificity of the enzyme towards a representative and structurally diverse range of methyl ester substrates, are described.

Authors
Toone, EJ; Werth, MJ; Jones, JB
MLA Citation
Toone, EJ, Werth, MJ, and Jones, JB. "Active-site model for interpreting and predicting the specificity of pig liver esterase." Journal of the American Chemical Society 112.12 (1990): 4946-4952.
Source
scival
Published In
Journal of the American Chemical Society
Volume
112
Issue
12
Publish Date
1990
Start Page
4946
End Page
4952

Computer Software Reviews

MLA Citation
"Computer Software Reviews." Journal of the American Chemical Society 111.5 (March 1989): 1942-1943.
Source
crossref
Published In
Journal of the American Chemical Society
Volume
111
Issue
5
Publish Date
1989
Start Page
1942
End Page
1943
DOI
10.1021/ja00187a600

Enzyme-catalyzed synthesis of carbohydrates

Authors
Toone, EJ; Simon, ES; Bednarski, MD; Whitesides, GM
MLA Citation
Toone, EJ, Simon, ES, Bednarski, MD, and Whitesides, GM. "Enzyme-catalyzed synthesis of carbohydrates." Tetrahedron 45.17 (1989): 5365-5422.
Source
scival
Published In
Tetrahedron
Volume
45
Issue
17
Publish Date
1989
Start Page
5365
End Page
5422

Preparation of cytidine 5'-monophospho-N-acetylneuraminic acid and uridine 5'-diphosphoglucuronic acid; syntheses of alpha-2, 6-sialyllactosamine, alpha-2, 6-sialyllactose, and hyaluronic acid.

Authors
Simon, ES; Toone, EJ; Ostroff, G; Bednarski, MD; Whitesides, GM
MLA Citation
Simon, ES, Toone, EJ, Ostroff, G, Bednarski, MD, and Whitesides, GM. "Preparation of cytidine 5'-monophospho-N-acetylneuraminic acid and uridine 5'-diphosphoglucuronic acid; syntheses of alpha-2, 6-sialyllactosamine, alpha-2, 6-sialyllactose, and hyaluronic acid." Methods Enzymol 179 (1989): 275-287.
PMID
2560122
Source
pubmed
Published In
Methods in Enzymology
Volume
179
Publish Date
1989
Start Page
275
End Page
287

ENZYMES IN ORGANIC SYNTHESIS. 42. INVESTIGATION OF THE EFFECTS OF THE ISOZYMAL COMPOSITION OF PIG LIVER ESTERASE ON ITS STEREOSELECTIVITY IN PREPARATIVE-SCALE ESTER HYDROLYSES OF ASYMMETRIC SYNTHETIC VALUE.

The stereospecificities of the isozyme components of commercially available pig liver esterase have been shown to be essentially the same toward representative monocyclic and acyclic diester substrates. This removes previous concerns that the isozymal composition of pig liver esterase, a widely used catalyst for chiral synthon production, might result in its not behaving consistently when applied as a catalyst for asymmetric synthetic purposes. The results establish that commercial pig liver esterase can be exploited synthetically as a chiral catalyst with confidence that it will behave as if it were a single protein.

Authors
Lam, LKP; Brown, CM; Jeso, BD; Lym, L; Toone, EJ; Jones, JB
MLA Citation
Lam, LKP, Brown, CM, Jeso, BD, Lym, L, Toone, EJ, and Jones, JB. "ENZYMES IN ORGANIC SYNTHESIS. 42. INVESTIGATION OF THE EFFECTS OF THE ISOZYMAL COMPOSITION OF PIG LIVER ESTERASE ON ITS STEREOSELECTIVITY IN PREPARATIVE-SCALE ESTER HYDROLYSES OF ASYMMETRIC SYNTHETIC VALUE." Journal of the American Chemical Society 110.13 (1988): 4409-4411.
Source
scival
Published In
Journal of the American Chemical Society
Volume
110
Issue
13
Publish Date
1988
Start Page
4409
End Page
4411

Determination of the absolute configuration of six-membered-ring ketones by 13C NMR

The diastereotopic splitting of the 13C NMR signals of the (2R,3R)-2,3-butanediol acetals of 39 chiral six-membered-ring ketones, including 2- and 3-substituted cyclohexanones, 2-alkyltetrahydropyran-4-ones, and 2- and 3-alkyltetrahydrothiopyran-4-ones, has been studied. The stereodifferentiation of the six-membered-ring-carbon atoms was shown to follow a very regular pattern, which is independent of substituents or of heteroatoms in the ring. Hence, an empirical rule could be deduced which allows the assignment of the absolute configuration of the original ketones from the 13C NMR spectrum of their acetals. The rule is only valid for six-membered-ring ketones having the chair form as the preferred conformation in the acetal. As shown for 11 examples, this rule is not valid for acyclic ketones, cyclopentanones, cycloheptanones, or cyclohexanones and cyclohexenones not having the chair form as the preferred conformation. The rule also permits the interpretation of the 13C NMR spectrum of the (2R,3R)-2,3-butanediol acetals of achiral cyclohexanones (seven examples). The stereodifferentiation is almost exclusively determined by the centers of chirality in the dioxolane ring. © 1987 American Chemical Society.

Authors
Lemière, GL; Dommisse, RA; Lepoivre, JA; Alderweireldt, FC; Hiemstra, H; Wynberg, H; Jones, JB; Toone, EJ
MLA Citation
Lemière, GL, Dommisse, RA, Lepoivre, JA, Alderweireldt, FC, Hiemstra, H, Wynberg, H, Jones, JB, and Toone, EJ. "Determination of the absolute configuration of six-membered-ring ketones by 13C NMR." Journal of the American Chemical Society 109.5 (1987): 1363-1370.
Source
scival
Published In
Journal of the American Chemical Society
Volume
109
Issue
5
Publish Date
1987
Start Page
1363
End Page
1370

Enzymes in organic synthesis. 40. Evaluation of the enantioselectivity of the pig liver esterase catalyzed hydrolyses of racemic piperidine carboxylic acid esters

Authors
Toone, EJ; Jones, JB
MLA Citation
Toone, EJ, and Jones, JB. "Enzymes in organic synthesis. 40. Evaluation of the enantioselectivity of the pig liver esterase catalyzed hydrolyses of racemic piperidine carboxylic acid esters." Canadian Journal of Chemistry 65.12 (1987): 2722-2726.
Source
scival
Published In
Canadian Journal of Chemistry
Volume
65
Issue
12
Publish Date
1987
Start Page
2722
End Page
2726

Photolysis of some ortho-methylated monochlorobiphenyls

A series of monochloro-o-methylated biphenyls was photolysed. Quantum yields of dechlorination lie in between the low values observed for 3- and 4-chlorobiphenyl and the high values obtained for 2-chlorobiphenyl. The results are interpreted in terms of raising the energy of the excited state due to steric crowding of the ortho-substituent and relief of strain upon the departure of an ortho-chlorine.

Authors
Bunce, NJ; Deschutter, CT; Toone, EJ
MLA Citation
Bunce, NJ, Deschutter, CT, and Toone, EJ. "Photolysis of some ortho-methylated monochlorobiphenyls." Journal of the Chemical Society, Perkin Transactions 2 6 (1983): 859-861.
Source
scival
Published In
Journal of the Chemical Society, Perkin Transactions 2
Issue
6
Publish Date
1983
Start Page
859
End Page
861

PHOTOCHEMISTRY OF BENZOPHENONE IN ACIDIFIED METHANOL

Authors
BUNCE, NJ; TOONE, EJ
MLA Citation
BUNCE, NJ, and TOONE, EJ. "PHOTOCHEMISTRY OF BENZOPHENONE IN ACIDIFIED METHANOL." JOURNAL OF CHEMICAL RESEARCH-S 5 (1983): 115-115.
Source
wos-lite
Published In
Journal of Chemical Research - Part S
Issue
5
Publish Date
1983
Start Page
115
End Page
115
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