You are here

McCafferty, Dewey G.

Overview:

Our research interests are broadly based in chemical biology, mechanistic enzymology and molecular medicine. Towards this end our group is engaged in understanding the chemical and kinetic mechanisms, substrate specificity and therapeutic importance of enzymes that posttranslationally modify chromatin, such as histone deacetylases, histone demethylases, histone methyl transferases, and chromatin assembly and remodeling complexes. Building on a mechanistic foundation, our laboratory is also interested in the design, chemical synthesis and evaluation of small molecules to modulate the activity of chromatin modifying enzymes within living cells. This work has recently led to the discovery of histone deamethylases as potential targets for anti-depression therapy. In addition, our laboratory also works to identify and develop novel strategies to overcome bacterial resistance to antibiotics through mechanistic characterization of enzymes involved in bacterial virulence, peptidoglycan biosynthesis, and teichoic acid biosynthesis. A central component of this research is the identification of novel anti-virulence chemotherapeutics and antibiotics capable of overcoming infections from antibiotic resistant bacteria. Our group also works to decode the molecular mechanisms of enzymes involved in mechanistically intriguing reactions from antibiotic natural product biosynthesis. Lastly, our group is working to develop a functional view of the molecular underpinning of initial signaling events in bacterial-induced inflammation, and in turn lay the foundation for the discovery and design of novel small molecule inhibitors of Crohn's disease, ulcerative colitis and related inflammatory bowel disorders.

Positions:

Professor of Chemistry

Chemistry
Trinity College of Arts & Sciences

Professor of Biochemistry

Biochemistry
School of Medicine

Faculty Network Member of The Energy Initiative

Duke University Energy Initiative
Institutes and Provost's Academic Units

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

B.S. 1990

B.S. — North Carolina State University

Ph.D. 1995

Ph.D. — University of North Carolina at Chapel Hill

News:

Grants:

Cell signaling through O-GlcNAc reader proteins

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
April 01, 2016
End Date
March 31, 2021

Enzymology of Chlamydial Pathogenesis

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2013
End Date
February 28, 2019

BC123577: Targeting Epigenetics Therapy for Estrogen Receptor Negative Breast Cancers

Administered By
Chemistry
AwardedBy
United States Army Medical Research and Materiel Command
Role
Principal Investigator
Start Date
September 30, 2013
End Date
September 29, 2016

Chemical Biology Approaches to Combat Parkinson's Disease and Dyskinesia

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2013
End Date
July 31, 2016

Automated detection of protein crystals in high-throughput crystallography experiments

Administered By
Duke Human Vaccine Institute
AwardedBy
North Carolina Biotechnology Center
Role
Major User
Start Date
April 01, 2014
End Date
April 30, 2015

Structural and Biochemical Studies of LpxC Inhibition

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

Bioorganic Mechanisms of Chromatin Modifying Enzymes

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
June 05, 2009
End Date
May 31, 2011

Bioorganic Mechanisms of Peptide Antibiotics

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
December 20, 2006
End Date
March 31, 2011

Mechanistic Analyses of Protein Deacetylation

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2006
End Date
February 28, 2008

RSF: Properties and Dependence Upon Histone Modifications

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
June 01, 2006
End Date
July 13, 2007
Show More

Awards:

Eli Lilly Award in Biological Chemistry. American Chemical Society.

Type
National
Awarded By
American Chemical Society
Date
January 01, 2005

Publications:

Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics.

We present protease specificity profiling based on quantitative proteomics in combination with proteome-derived peptide libraries. Peptide libraries are generated by endoproteolytic digestion of proteomes without chemical modification of primary amines before exposure to a protease under investigation. After incubation with a test protease, treated and control libraries are differentially isotope-labeled using cost-effective reductive dimethylation. Upon analysis by liquid chromatography-tandem mass spectrometry, cleavage products of the test protease appear as semi-specific peptides that are enriched for the corresponding isotope label. We validate our workflow with two proteases with well-characterized specificity profiles: trypsin and caspase-3. We provide the first specificity profile of a protease encoded by a human endogenous retrovirus and for chlamydial protease-like activity factor (CPAF). For CPAF, we also highlight the structural basis of negative subsite cooperativity between subsites S1 and S2'. For A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -4, -5, and -15, we show a canonical preference profile, including glutamate in P1 and glycine in P3'. In total, we report nearly 4000 cleavage sites for seven proteases. Our protocol is fast, avoids enrichment or synthesis steps, and enables probing for lysine selectivity as well as subsite cooperativity. Due to its simplicity, we anticipate usability by most proteomic laboratories.

Authors
Biniossek, ML; Niemer, M; Maksimchuk, K; Mayer, B; Fuchs, J; Huesgen, PF; McCafferty, DG; Turk, B; Fritz, G; Mayer, J; Haecker, G; Mach, L; Schilling, O
MLA Citation
Biniossek, ML, Niemer, M, Maksimchuk, K, Mayer, B, Fuchs, J, Huesgen, PF, McCafferty, DG, Turk, B, Fritz, G, Mayer, J, Haecker, G, Mach, L, and Schilling, O. "Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics." Molecular & cellular proteomics : MCP 15.7 (July 2016): 2515-2524.
PMID
27122596
Source
epmc
Published In
Molecular & cellular proteomics : MCP
Volume
15
Issue
7
Publish Date
2016
Start Page
2515
End Page
2524
DOI
10.1074/mcp.o115.056671

Lysine-Specific Demethylase 1A (KDM1A/LSD1): Product Recognition and Kinetic Analysis of Full-Length Histones.

Lysine-specific demethylase 1A (KDM1A/LSD1) is a FAD-dependent enzyme that catalyzes the oxidative demethylation of histone H3K4me1/2 and H3K9me1/2 repressing and activating transcription, respectively. Although the active site is expanded compared to that of members of the greater amine oxidase superfamily, it is too sterically restricted to encompass the minimal 21-mer peptide substrate footprint. The remainder of the substrate/product is therefore expected to extend along the surface of KDM1A. We show that full-length histone H3, which lacks any posttranslational modifications, is a tight-binding, competitive inhibitor of KDM1A demethylation activity with a Ki of 18.9 ± 1.2 nM, a value that is approximately 100-fold higher than that of the 21-mer peptide product. The relative H3 affinity is independent of preincubation time, suggesting that H3 rapidly reaches equilibrium with KDM1A. Jump dilution experiments confirmed the increased binding affinity of full-length H3 was at least partially due to a slow off rate (koff) of 1.2 × 10(-3) s(-1), corresponding to a half-life (t1/2) of 9.63 min, and a residence time (τ) of 13.9 min. Independent affinity capture surface plasmon resonance experiments confirmed the tight-binding nature of the H3/KDM1A interaction, revealing a Kd of 9.02 ± 2.3 nM, a kon of (9.3 ± 1.5) × 10(4) M(-1) s(-1), and a koff of (8.4 ± 0.3) × 10(-4) s(-1). Additionally, no other core histones exhibited inhibition of KDM1A demethylation activity, which is consistent with H3 being the preferred histone substrate of KDM1A versus H2A, H2B, and H4. Together, these data suggest that KDM1A likely contains a histone H3 secondary specificity element on the enzyme surface that contributes significantly to its recognition of substrates and products.

Authors
Burg, JM; Gonzalez, JJ; Maksimchuk, KR; McCafferty, DG
MLA Citation
Burg, JM, Gonzalez, JJ, Maksimchuk, KR, and McCafferty, DG. "Lysine-Specific Demethylase 1A (KDM1A/LSD1): Product Recognition and Kinetic Analysis of Full-Length Histones." Biochemistry 55.11 (March 2016): 1652-1662.
PMID
26673564
Source
epmc
Published In
Biochemistry
Volume
55
Issue
11
Publish Date
2016
Start Page
1652
End Page
1662
DOI
10.1021/acs.biochem.5b01135

The Chlamydia trachomatis Protease CPAF Contains a Cryptic PDZ-Like Domain with Similarity to Human Cell Polarity and Tight Junction PDZ-Containing Proteins.

The need for more effective anti-chlamydial therapeutics has sparked research efforts geared toward further understanding chlamydial pathogenesis mechanisms. Recent studies have implicated the secreted chlamydial serine protease, chlamydial protease-like activity factor (CPAF) as potentially important for chlamydial pathogenesis. By mechanisms that remain to be elucidated, CPAF is directed to a discrete group of substrates, which are subsequently cleaved or degraded. While inspecting the previously solved CPAF crystal structure, we discovered that CPAF contains a cryptic N-terminal PSD95 Dlg ZO-1 (PDZ) domain spanning residues 106-212 (CPAF106-212). This PDZ domain is unique in that it bears minimal sequence similarity to canonical PDZ-forming sequences and displays little sequence and structural similarity to known chlamydial PDZ domains. We show that the CPAF106-212 sequence is homologous to PDZ domains of human tight junction proteins.

Authors
Maksimchuk, KR; Alser, KA; Mou, R; Valdivia, RH; McCafferty, DG
MLA Citation
Maksimchuk, KR, Alser, KA, Mou, R, Valdivia, RH, and McCafferty, DG. "The Chlamydia trachomatis Protease CPAF Contains a Cryptic PDZ-Like Domain with Similarity to Human Cell Polarity and Tight Junction PDZ-Containing Proteins." PloS one 11.2 (January 2016): e0147233-.
PMID
26829550
Source
epmc
Published In
PloS one
Volume
11
Issue
2
Publish Date
2016
Start Page
e0147233
DOI
10.1371/journal.pone.0147233

A rationally-designed chimeric KDM1A/KDM1B histone demethylase tower domain deletion mutant retaining enzymatic activity.

A target with therapeutic potential, lysine-specific demethylase 1A (KDM1A) is a regulator of gene expression whose tower domain is a protein-protein interaction motif. This domain facilitates the interaction of KDM1A with coregulators and multiprotein complexes that direct its activity to nucleosomes. We describe the design and characterization of a chimeric 'towerless' KDM1A, termed nΔ150 KDM1AΔTower KDM1B chimera (chKDM1AΔTower), which incorporates a region from the paralog lysine-specific demethylase 1B (KDM1B). This chimera copurifies with FAD and displays demethylase activity, but fails to bind the partner protein corepressor of the RE1-silencing transcription factor (CoREST). We conclude that KDM1A catalysis can be decoupled from tower-dependent interactions, lending chKDM1AΔTower useful for dissecting molecular contributions to KDM1A function.

Authors
Burg, JM; Makhoul, AT; Pemble, CW; Link, JE; Heller, FJ; McCafferty, DG
MLA Citation
Burg, JM, Makhoul, AT, Pemble, CW, Link, JE, Heller, FJ, and McCafferty, DG. "A rationally-designed chimeric KDM1A/KDM1B histone demethylase tower domain deletion mutant retaining enzymatic activity." FEBS letters 589.18 (August 2015): 2340-2346.
PMID
26226427
Source
epmc
Published In
FEBS Letters
Volume
589
Issue
18
Publish Date
2015
Start Page
2340
End Page
2346
DOI
10.1016/j.febslet.2015.07.028

KDM1 class flavin-dependent protein lysine demethylases.

Flavin-dependent, lysine-specific protein demethylases (KDM1s) are a subfamily of amine oxidases that catalyze the selective posttranslational oxidative demethylation of methyllysine side chains within protein and peptide substrates. KDM1s participate in the widespread epigenetic regulation of both normal and disease state transcriptional programs. Their activities are central to various cellular functions, such as hematopoietic and neuronal differentiation, cancer proliferation and metastasis, and viral lytic replication and establishment of latency. Interestingly, KDM1s function as catalytic subunits within complexes with coregulatory molecules that modulate enzymatic activity of the demethylases and coordinate their access to specific substrates at distinct sites within the cell and chromatin. Although several classes of KDM1-selective small molecule inhibitors have been recently developed, these pan-active site inhibition strategies lack the ability to selectively discriminate between KDM1 activity in specific, and occasionally opposing, functional contexts within these complexes. Here we review the discovery of this class of demethylases, their structures, chemical mechanisms, and specificity. Additionally, we review inhibition of this class of enzymes as well as emerging interactions with coregulatory molecules that regulate demethylase activity in highly specific functional contexts of biological and potential therapeutic importance.

Authors
Burg, JM; Link, JE; Morgan, BS; Heller, FJ; Hargrove, AE; McCafferty, DG
MLA Citation
Burg, JM, Link, JE, Morgan, BS, Heller, FJ, Hargrove, AE, and McCafferty, DG. "KDM1 class flavin-dependent protein lysine demethylases." Biopolymers 104.4 (July 2015): 213-246. (Review)
PMID
25787087
Source
epmc
Published In
Biopolymers
Volume
104
Issue
4
Publish Date
2015
Start Page
213
End Page
246
DOI
10.1002/bip.22643

Reassessing the role of the secreted protease CPAF in Chlamydia trachomatis infection through genetic approaches.

The secreted Chlamydia protease CPAF cleaves a defined set of mammalian and Chlamydia proteins in vitro. As a result, this protease has been proposed to modulate a range of bacterial and host cellular functions. However, it has recently come into question the extent to which many of its identified substrates constitute bona fide targets of proteolysis in infected host cell rather than artifacts of postlysis degradation. Here, we clarify the role played by CPAF in cellular models of infection by analyzing Chlamydia trachomatis mutants deficient for CPAF activity. Using reverse genetic approaches, we identified two C. trachomatis strains possessing nonsense, loss-of-function mutations in cpa (CT858) and a third strain containing a mutation in type II secretion (T2S) machinery that inhibited CPAF activity by blocking zymogen secretion and subsequent proteolytic maturation into the active hydrolase. HeLa cells infected with T2S(-) or CPAF(-) C. trachomatis mutants lacked detectable in vitro CPAF proteolytic activity and were not defective for cellular traits that have been previously attributed to CPAF activity, including resistance to staurosporine-induced apoptosis, Golgi fragmentation, altered NFκB-dependent gene expression, and resistance to reinfection. However, CPAF-deficient mutants did display impaired generation of infectious elementary bodies (EBs), indicating an important role for this protease in the full replicative potential of C. trachomatis. In addition, we provide compelling evidence in live cells that CPAF-mediated protein processing of at least two host protein targets, vimentin filaments and the nuclear envelope protein lamin-associated protein-1 (LAP1), occurs rapidly after the loss of the inclusion membrane integrity, but before loss of plasma membrane permeability and cell lysis. CPAF-dependent processing of host proteins correlates with a loss of inclusion membrane integrity, and so we propose that CPAF plays a role late in infection, possibly during the stages leading to the dismantling of the infected cell prior to the release of EBs during cell lysis.

Authors
Snavely, EA; Kokes, M; Dunn, JD; Saka, HA; Nguyen, BD; Bastidas, RJ; McCafferty, DG; Valdivia, RH
MLA Citation
Snavely, EA, Kokes, M, Dunn, JD, Saka, HA, Nguyen, BD, Bastidas, RJ, McCafferty, DG, and Valdivia, RH. "Reassessing the role of the secreted protease CPAF in Chlamydia trachomatis infection through genetic approaches." Pathogens and disease 71.3 (August 2014): 336-351.
PMID
24838663
Source
epmc
Published In
Pathogens and Disease
Volume
71
Issue
3
Publish Date
2014
Start Page
336
End Page
351
DOI
10.1111/2049-632x.12179

Sortase-catalyzed initiator attachment enables high yield growth of a stealth polymer from the C terminus of a protein.

Conventional methods for synthesizing protein/peptide-polymer conjugates, as a means to improve the pharmacological properties of therapeutic biomolecules, typically have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of site- specificity, which results in heterogeneous products with significantly compromised bioactivity. To address these limitations, the use of sortase A from Staphylococcus aureus is demonstrated to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein (GFP), followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeds with high specificity and near-complete (≈95%) product conversion. Subsequent in situ ATRP in aqueous buffer produces 1:1 stoichiometric conjugates with >90% yield, low dispersity, and no denaturation of the protein. This approach introduces a simple and useful method for high yield synthesis of protein/peptide-polymer conjugates.

Authors
Qi, Y; Amiram, M; Gao, W; McCafferty, DG; Chilkoti, A
MLA Citation
Qi, Y, Amiram, M, Gao, W, McCafferty, DG, and Chilkoti, A. "Sortase-catalyzed initiator attachment enables high yield growth of a stealth polymer from the C terminus of a protein." Macromol Rapid Commun 34.15 (August 2013): 1256-1260.
PMID
23836349
Source
pubmed
Published In
Macromolecular Rapid Communications
Volume
34
Issue
15
Publish Date
2013
Start Page
1256
End Page
1260
DOI
10.1002/marc.201300460

Three-in-one chromatography-free purification, tag removal, and site-specific modification of recombinant fusion proteins using sortase A and elastin-like polypeptides.

Authors
Bellucci, JJ; Amiram, M; Bhattacharyya, J; McCafferty, D; Chilkoti, A
MLA Citation
Bellucci, JJ, Amiram, M, Bhattacharyya, J, McCafferty, D, and Chilkoti, A. "Three-in-one chromatography-free purification, tag removal, and site-specific modification of recombinant fusion proteins using sortase A and elastin-like polypeptides." Angew Chem Int Ed Engl 52.13 (March 25, 2013): 3703-3708.
PMID
23424160
Source
pubmed
Published In
Angewandte Chemie International Edition
Volume
52
Issue
13
Publish Date
2013
Start Page
3703
End Page
3708
DOI
10.1002/anie.201208292

Sortase-Catalyzed Initiator Attachment Enables High Yield Growth of a Stealth Polymer from the C Terminus of a Protein

Conventional methods for synthesizing protein/peptide-polymer conjugates, as a means to improve the pharmacological properties of therapeutic biomolecules, typically have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of site- specificity, which results in heterogeneous products with significantly compromised bioactivity. To address these limitations, the use of sortase A from Staphylococcus aureus is demonstrated to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein (GFP), followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeds with high specificity and near-complete (≈95%) product conversion. Subsequent in situ ATRP in aqueous buffer produces 1:1 stoichiometric conjugates with >90% yield, low dispersity, and no denaturation of the protein. This approach introduces a simple and useful method for high yield synthesis of protein/peptide-polymer conjugates. A general method is developed for the high-yield synthesis of C-terminally site-specific and one-to-one stoichiometric protein/peptide-polymer conjugates. Sortase A from Staphylococcus aureus is used to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein, followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Authors
Qi, Y; Amiram, M; Gao, W; McCafferty, DG; Chilkoti, A
MLA Citation
Qi, Y, Amiram, M, Gao, W, McCafferty, DG, and Chilkoti, A. "Sortase-Catalyzed Initiator Attachment Enables High Yield Growth of a Stealth Polymer from the C Terminus of a Protein." Macromolecular Rapid Communications 34.15 (2013): 1256-1260.
Source
scival
Published In
Macromolecular Rapid Communications
Volume
34
Issue
15
Publish Date
2013
Start Page
1256
End Page
1260
DOI
10.1002/marc.201300460

Sortases

Authors
McCafferty, DG; Melvin, JA
MLA Citation
McCafferty, DG, and Melvin, JA. "Sortases." Handbook of Proteolytic Enzymes 2 (2013): 2459-2465.
Source
scival
Published In
Handbook of Proteolytic Enzymes
Volume
2
Publish Date
2013
Start Page
2459
End Page
2465
DOI
10.1016/B978-0-12-382219-2.00549-4

Monitoring β-secretase activity in living cells with a membrane-anchored FRET probe.

Authors
Folk, DS; Torosian, JC; Hwang, S; McCafferty, DG; Franz, KJ
MLA Citation
Folk, DS, Torosian, JC, Hwang, S, McCafferty, DG, and Franz, KJ. "Monitoring β-secretase activity in living cells with a membrane-anchored FRET probe." Angew Chem Int Ed Engl 51.43 (October 22, 2012): 10795-10799.
PMID
23023944
Source
pubmed
Published In
Angewandte Chemie International Edition
Volume
51
Issue
43
Publish Date
2012
Start Page
10795
End Page
10799
DOI
10.1002/anie.201206673

Lysine-specific histone demethylase 1 inhibitors control breast cancer proliferation in ERα-dependent and -independent manners.

Lysine specific demethylase 1 (LSD1, also known as KDM1) is a histone modifying enzyme that regulates the expression of many genes important in cancer progression and proliferation. It is present in various transcriptional complexes including those containing the estrogen receptor (ER). Indeed, inhibition of LSD1 activity and or expression has been shown to attenuate estrogen signaling in breast cancer cells in vitro, implicating this protein in the pathogenesis of cancer. Herein we describe experiments that utilize small molecule inhibitors, phenylcyclopropylamines, along with small interfering RNA to probe the role of LSD1 in breast cancer proliferation and in estrogen-dependent gene transcription. Surprisingly, whereas we have confirmed that inhibition of LSD1 strongly inhibits proliferation of breast cancer cells, we have determined that the cytostatic actions of LSD1 inhibition are not impacted by ER status. These data suggest that LSD1 may be a useful therapeutic target in several types of breast cancer; most notably, inhibitors of LSD1 may have utility in the treatment of ER-negative cancers for which there are minimal therapeutic options.

Authors
Pollock, JA; Larrea, MD; Jasper, JS; McDonnell, DP; McCafferty, DG
MLA Citation
Pollock, JA, Larrea, MD, Jasper, JS, McDonnell, DP, and McCafferty, DG. "Lysine-specific histone demethylase 1 inhibitors control breast cancer proliferation in ERα-dependent and -independent manners." ACS Chem Biol 7.7 (July 20, 2012): 1221-1231.
PMID
22533360
Source
pubmed
Published In
ACS Chemical Biology
Volume
7
Issue
7
Publish Date
2012
Start Page
1221
End Page
1231
DOI
10.1021/cb300108c

Studies on the biosynthesis of the lipodepsipeptide antibiotic Ramoplanin A2.

Ramoplanin, a non-ribosomally synthesized peptide antibiotic, is highly effective against several drug-resistant Gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), two important opportunistic human pathogens. Recently, the biosynthetic cluster from the ramoplanin producer Actinoplanes ATCC 33076 was sequenced, revealing an unusual architecture of fatty acid and non-ribosomal peptide synthetase biosynthetic genes (NRPSs). The first steps towards understanding how these biosynthetic enzymes cooperatively interact to produce the depsipeptide product are expression and isolation of each enzyme to probe its specificity and function. Here we describe the successful production of soluble enzymes from within the ramoplanin locus and the confirmation of their specific role in biosynthesis. These methods may be broadly applicable to the production of biosynthetic enzymes from other natural product biosynthetic gene clusters, especially those that have been refractory to production in heterologous hosts despite standard expression optimization methods.

Authors
Hoertz, AJ; Hamburger, JB; Gooden, DM; Bednar, MM; McCafferty, DG
MLA Citation
Hoertz, AJ, Hamburger, JB, Gooden, DM, Bednar, MM, and McCafferty, DG. "Studies on the biosynthesis of the lipodepsipeptide antibiotic Ramoplanin A2." Bioorg Med Chem 20.2 (January 15, 2012): 859-865.
PMID
22222159
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry
Volume
20
Issue
2
Publish Date
2012
Start Page
859
End Page
865
DOI
10.1016/j.bmc.2011.11.062

Proton-coupled electron transfer

Proton-coupled electron transfer (PCET) has a major role in chemistry and biology and its implications for catalysis and energy conversion. Photosynthesis is a spectacular example of PCET in action with the transfer of 24 e- and 24 H+ driven by at least 48 photons. In PCET half reactions, variations in pH influence driving force. Both HAT (H-atom transfer) and EPT (electron-proton transfer) are elementary steps by which PCET reactions can occur. In H-atom transfer (HAT), both the transferring electron and proton come from the same bond in one of the reactants. Multiple Site Electron-Proton Transfer (MS-EPT) is microscopically more complex than electron or proton transfer. It shares with electron transfer requirements for medium and intramolecular reorganization but with the additional complexity of a coupled proton transfer. PCET plays a critical role in many enzymatic pathways that control life such as photosynthesis, respiration, and DNA repair.

Authors
Weinberg, DR; Gagliardi, CJ; Hull, JF; Murphy, CF; Kent, CA; Westlake, BC; Paul, A; Ess, DH; McCafferty, DG; Meyer, TJ
MLA Citation
Weinberg, DR, Gagliardi, CJ, Hull, JF, Murphy, CF, Kent, CA, Westlake, BC, Paul, A, Ess, DH, McCafferty, DG, and Meyer, TJ. "Proton-coupled electron transfer." Chemical Reviews 112.7 (2012): 4016-4093.
PMID
22702235
Source
scival
Published In
Chemical Reviews
Volume
112
Issue
7
Publish Date
2012
Start Page
4016
End Page
4093
DOI
10.1021/cr200177j

Staphylococcus aureus sortase A contributes to the Trojan horse mechanism of immune defense evasion with its intrinsic resistance to Cys184 oxidation.

Staphylococcus aureus is a Gram-positive bacterial pathogen that causes serious infections which have become increasingly difficult to treat due to antimicrobial resistance and natural virulence strategies. Bacterial sortase enzymes are important virulence factors and good targets for future antibiotic development. It has recently been shown that sortase enzymes are integral to bacterial survival of phagocytosis, an underappreciated, but vital, step in S. aureus pathogenesis. Of note, the reaction mechanism of sortases relies on a solvent-accessible cysteine for transpeptidation. Because of the common strategy of oxidative damage employed by professional phagocytes to kill pathogens, it is possible that this cysteine may be oxidized inside the phagosome, thereby inhibiting the enzyme. This study addresses this apparent paradox by assessing the ability of physiological reactive oxygen species, hydrogen peroxide and hypochlorite, to inhibit sortase A (SrtA) from S. aureus. Surprisingly, we found that SrtA is highly resistant to oxidative inhibition, both in vitro and in vivo. The mechanism of resistance to oxidative damage is likely mediated by maintaining a high reduction potential of the catalytic cysteine residue, Cys184. This is due to the unusual active site utilized by S. aureus SrtA, which employs a reverse protonation mechanism for transpeptidation, resulting in a high pK(a) as well as reduction potential for Cys184. The results of this study suggest that S. aureus SrtA is able to withstand the extreme conditions encountered in the phagosome and maintain function, contributing to survival of phagocytotic killing.

Authors
Melvin, JA; Murphy, CF; Dubois, LG; Thompson, JW; Moseley, MA; McCafferty, DG
MLA Citation
Melvin, JA, Murphy, CF, Dubois, LG, Thompson, JW, Moseley, MA, and McCafferty, DG. "Staphylococcus aureus sortase A contributes to the Trojan horse mechanism of immune defense evasion with its intrinsic resistance to Cys184 oxidation." Biochemistry 50.35 (September 6, 2011): 7591-7599.
PMID
21812416
Source
pubmed
Published In
Biochemistry
Volume
50
Issue
35
Publish Date
2011
Start Page
7591
End Page
7599
DOI
10.1021/bi200844h

Chlamydia protease-like activity factor (CPAF): characterization of proteolysis activity in vitro and development of a nanomolar affinity CPAF zymogen-derived inhibitor.

During infection of epithelial cells, the obligate intracellular pathogen Chlamydia trachomatis secretes the serine protease Chlamydia protease-like activity factor (CPAF) into the host cytosol to regulate a range of host cellular processes through targeted proteolysis. Here we report the development of an in vitro assay for the enzyme and the discovery of a cell-permeable CPAF zymogen-based peptide inhibitor with nanomolar inhibitory affinity. Treating C. trachomatis-infected HeLa cells with this inhibitor prevented CPAF cleavage of the intermediate filament vimentin and led to the loss of vimentin cage surrounding the intracellular vacuole. Because Chlamydia is a genetically intractable organism, this inhibitor may serve as a tool for understanding the role of CPAF in pathogenesis.

Authors
Bednar, MM; Jorgensen, I; Valdivia, RH; McCafferty, DG
MLA Citation
Bednar, MM, Jorgensen, I, Valdivia, RH, and McCafferty, DG. "Chlamydia protease-like activity factor (CPAF): characterization of proteolysis activity in vitro and development of a nanomolar affinity CPAF zymogen-derived inhibitor." Biochemistry 50.35 (September 6, 2011): 7441-7443.
PMID
21830778
Source
pubmed
Published In
Biochemistry
Volume
50
Issue
35
Publish Date
2011
Start Page
7441
End Page
7443
DOI
10.1021/bi201098r

The Chlamydia protease CPAF regulates host and bacterial proteins to maintain pathogen vacuole integrity and promote virulence.

The obligate intracellular bacterial pathogen Chlamydia trachomatis injects numerous effector proteins into the epithelial cell cytoplasm to manipulate host functions important for bacterial survival. In addition, the bacterium secretes a serine protease, chlamydial protease-like activity factor (CPAF). Although several CPAF targets are reported, the significance of CPAF-mediated proteolysis is unclear due to the lack of specific CPAF inhibitors and the diversity of host targets. We report that CPAF also targets chlamydial effectors secreted early during the establishment of the pathogen-containing vacuole ("inclusion"). We designed a cell-permeable CPAF-specific inhibitory peptide and used it to determine that CPAF prevents superinfection by degrading early Chlamydia effectors translocated during entry into a preinfected cell. Prolonged CPAF inhibition leads to loss of inclusion integrity and caspase-1-dependent death of infected epithelial cells. Thus, CPAF functions in niche protection, inclusion integrity and pathogen survival, making the development of CPAF-specific protease inhibitors an attractive antichlamydial therapeutic strategy.

Authors
Jorgensen, I; Bednar, MM; Amin, V; Davis, BK; Ting, JPY; McCafferty, DG; Valdivia, RH
MLA Citation
Jorgensen, I, Bednar, MM, Amin, V, Davis, BK, Ting, JPY, McCafferty, DG, and Valdivia, RH. "The Chlamydia protease CPAF regulates host and bacterial proteins to maintain pathogen vacuole integrity and promote virulence." Cell Host Microbe 10.1 (July 21, 2011): 21-32.
PMID
21767809
Source
pubmed
Published In
Cell Host and Microbe
Volume
10
Issue
1
Publish Date
2011
Start Page
21
End Page
32
DOI
10.1016/j.chom.2011.06.008

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

Autocatalytic intramolecular isopeptide bond formation in gram-positive bacterial pili: a QM/MM simulation.

Many gram-positive pathogens possess external pili or fimbriae with which they adhere to host cells during the infection process. Unusual dual intramolecular isopeptide bonds between Asn and Lys side chains within the N-terminal and C-terminal domains of the pilus subunits have been observed initially in the Streptococcus pyogenes pilin subunit Spy0128 and subsequently in GBS52 from Streptococcus agalactiae, in the BcpA major pilin of Bacillus cereus and in the RrgB pilin of Streptococcus pneumoniae, among others. Within each pilin subunit, intramolecular isopeptide bonds serve to stabilize the protein. These bonds provide a means to withstand large external mechanical forces, as well as possibly assisting in supporting a conformation favored for pilin subunit polymerization via sortase transpeptidases. Genome-wide analyses of pili-containing gram-positive bacteria are known or suspected to contain isopeptide bonds in pilin subunits. For the autocatalytic formation of isopeptide cross-links, a conservation of three amino acids including Asn, Lys, and a catalytically important acidic Glu (or Asp) residue are responsible. However, the chemical mechanism of how isopeptide bonds form within pilin remains poorly understood. Although it is possible that several mechanistic paths could lead to isopeptide bond formation in pili, the requirement of a conserved glutamate and highly organized positioning of residues within the hydrophobic environment of the active site were found in numerous pilin crystal structures such as Spy0128 and RrgB. This suggests a mechanism involving direct coupling of lysine side chain amine to the asparagine carboxamide mediated by critical acid/base or hydrogen bonding interactions with the catalytic glutamate residue. From this mechanistic perspective, we used the QM/MM minimum free-energy path method to examine the reaction details of forming the isopeptide bonds with Spy0128 as a model pilin, specifically focusing on the role of the glutamate in catalysis. It was determined that the reaction mechanism likely consists of two major steps: the nucleophilic attack on Cγ by nitrogen in the unprotonated Lys ε-amino group and, then two concerted proton transfers occur during the formation of the intramolecular isopeptide bond to subsequently release ammonia. More importantly, within the dual active sites of Spy0128, Glu(117), and Glu(258) residues function as crucial catalysts for each isopeptide bond formation, respectively, by relaying two proton transfers. This work also suggests that domain-domain interactions within Spy0128 may modulate the reactivity of residues within each active site. Our results may hopefully shed light on the molecular mechanisms of pilin biogenesis in gram-positive bacteria.

Authors
Hu, X; Hu, H; Melvin, JA; Clancy, KW; McCafferty, DG; Yang, W
MLA Citation
Hu, X, Hu, H, Melvin, JA, Clancy, KW, McCafferty, DG, and Yang, W. "Autocatalytic intramolecular isopeptide bond formation in gram-positive bacterial pili: a QM/MM simulation." J Am Chem Soc 133.3 (January 26, 2011): 478-485.
PMID
21142157
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
133
Issue
3
Publish Date
2011
Start Page
478
End Page
485
DOI
10.1021/ja107513t

Found in translation: Applications of protein and peptide molecular diversity

Authors
Cropp, TA; McCafferty, DG
MLA Citation
Cropp, TA, and McCafferty, DG. "Found in translation: Applications of protein and peptide molecular diversity." Current Opinion in Chemical Biology 15.3 (2011): 347-349.
PMID
21602093
Source
scival
Published In
Current Opinion in Chemical Biology
Volume
15
Issue
3
Publish Date
2011
Start Page
347
End Page
349
DOI
10.1016/j.cbpa.2011.04.013

Generation of ramoplanin-resistant Staphylococcus aureus.

Ramoplanin is a lipoglycodepsipeptide antimicrobial active against clinically important Gram-positive bacteria including methicillin-resistant Staphylococcus aureus. To proactively examine ramoplanin resistance, we subjected S. aureus NCTC 8325-4 to serial passage in the presence of increasing concentrations of ramoplanin, generating the markedly resistant strain RRSA16. Susceptibility testing of RRSA16 revealed the unanticipated acquisition of cross-resistance to vancomycin and nisin. RRSA16 displayed phenotypes, including a thickened cell wall and reduced susceptibility to Triton X-100-induced autolysis, which are associated with vancomycin intermediate-resistant S. aureus strains. Passage of RRSA16 for 18 days in a drug-free medium yielded strain R16-18d with restored antibiotic susceptibility. The RRSA16 isolate may be used to identify the genetic and biochemical basis for ramoplanin resistance and to further our understanding of the evolution of antibiotic cross-resistance mechanisms in S. aureus.

Authors
Schmidt, JW; Greenough, A; Burns, M; Luteran, AE; McCafferty, DG
MLA Citation
Schmidt, JW, Greenough, A, Burns, M, Luteran, AE, and McCafferty, DG. "Generation of ramoplanin-resistant Staphylococcus aureus." FEMS Microbiol Lett 310.2 (September 1, 2010): 104-111.
PMID
20659164
Source
pubmed
Published In
Fems Microbiology Letters
Volume
310
Issue
2
Publish Date
2010
Start Page
104
End Page
111
DOI
10.1111/j.1574-6968.2010.02051.x

Sortase transpeptidases: insights into mechanism, substrate specificity, and inhibition.

Gram-positive bacteria pose a serious healthcare threat. The growing antibiotic resistance epidemic creates a dire need for new antibiotic targets. The sortase family of enzymes is a promising target for antimicrobial therapy. This review covers the current knowledge of the mechanism, substrate specificity, and inhibitory studies of the Gram-positive bacterial [corrected] enzyme sortase.

Authors
Clancy, KW; Melvin, JA; McCafferty, DG
MLA Citation
Clancy, KW, Melvin, JA, and McCafferty, DG. "Sortase transpeptidases: insights into mechanism, substrate specificity, and inhibition." Biopolymers 94.4 (2010): 385-396. (Review)
PMID
20593474
Source
pubmed
Published In
Biopolymers
Volume
94
Issue
4
Publish Date
2010
Start Page
385
End Page
396
DOI
10.1002/bip.21472

A crystal structure of a dimer of the antibiotic ramoplanin illustrates membrane positioning and a potential Lipid II docking interface.

The glycodepsipeptide antibiotic ramoplanin A2 is in late stage clinical development for the treatment of infections from Gram-positive pathogens, especially those that are resistant to first line antibiotics such as vancomycin. Ramoplanin A2 achieves its antibacterial effects by interfering with production of the bacterial cell wall; it indirectly inhibits the transglycosylases responsible for peptidoglycan biosynthesis by sequestering their Lipid II substrate. Lipid II recognition and sequestration occur at the interface between the extracellular environment and the bacterial membrane. Therefore, we determined the structure of ramoplanin A2 in an amphipathic environment, using detergents as membrane mimetics, to provide the most physiologically relevant structural context for mechanistic and pharmacological studies. We report here the X-ray crystal structure of ramoplanin A2 at a resolution of 1.4 A. This structure reveals that ramoplanin A2 forms an intimate and highly amphipathic dimer and illustrates the potential means by which it interacts with bacterial target membranes. The structure also suggests a mechanism by which ramoplanin A2 recognizes its Lipid II ligand.

Authors
Hamburger, JB; Hoertz, AJ; Lee, A; Senturia, RJ; McCafferty, DG; Loll, PJ
MLA Citation
Hamburger, JB, Hoertz, AJ, Lee, A, Senturia, RJ, McCafferty, DG, and Loll, PJ. "A crystal structure of a dimer of the antibiotic ramoplanin illustrates membrane positioning and a potential Lipid II docking interface." Proc Natl Acad Sci U S A 106.33 (August 18, 2009): 13759-13764.
PMID
19666597
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
33
Publish Date
2009
Start Page
13759
End Page
13764
DOI
10.1073/pnas.0904686106

Critical role of NOD2 in regulating the immune response to Staphylococcus aureus.

NOD2 (the nucleotide-binding oligomerization domain containing protein 2) is known to be involved in host recognition of bacteria, although its role in the host response to Staphylococcus aureus infection is unknown. NOD2-deficient (Nod2(-/-)) mice and wild-type (WT) littermate controls were injected intraperitoneally with S. aureus suspension (10(7) bacteria/g of body weight), and their survival was monitored. Cultured bone marrow-derived neutrophils were harvested from Nod2(-/-) and WT mice and tested for cytokine production and phagocytosis. Compared to WT mice, Nod2(-/-) mice were significantly more susceptible to S. aureus infection (median survival of 1.5 days versus >5 days; P = 0.003) and had a significantly higher bacterial tissue burden. Cultured bone marrow-derived neutrophils from Nod2(-/-) and WT mice had similar levels of peritoneal neutrophil recruitment and intracellular killing, but bone marrow-derived neutrophils from Nod2(-/-) mice had significantly reduced ability to internalize fluorescein-labeled S. aureus. Nod2(-/-) mice had significantly higher levels of Th1-derived cytokines in serum (tumor necrosis factor alpha, gamma interferon, and interleukin-2 [IL-2]) compared to WT mice, whereas the levels of Th2-derived cytokines (IL-1beta, IL-4, IL-6, and IL-10) were similar in Nod2(-/-) and WT mice. Thus, mice deficient in NOD2 are more susceptible to S. aureus. Increased susceptibility is due in part to defective neutrophil phagocytosis, elevated serum levels of Th1 cytokines, and a higher bacterial tissue burden.

Authors
Deshmukh, HS; Hamburger, JB; Ahn, SH; McCafferty, DG; Yang, SR; Fowler, VG
MLA Citation
Deshmukh, HS, Hamburger, JB, Ahn, SH, McCafferty, DG, Yang, SR, and Fowler, VG. "Critical role of NOD2 in regulating the immune response to Staphylococcus aureus." Infect Immun 77.4 (April 2009): 1376-1382.
PMID
19139201
Source
pubmed
Published In
Infection and immunity
Volume
77
Issue
4
Publish Date
2009
Start Page
1376
End Page
1382
DOI
10.1128/IAI.00940-08

Use of pH and kinetic isotope effects to establish chemistry as rate-limiting in oxidation of a peptide substrate by LSD1

The mechanism of oxidation of a peptide substrate by the flavoprotein lysine-specific demethylase (LSD1) has been examined using the effects of pH and isotopic substitution on steady-state and rapid-reaction kinetic parameters. The substrate contained the 21 N-terminal residues of histone H3, with a dimethylated lysyl residue at position 4. At pH 7.5, the rate constant for flavin reduction, kred, equals kcat, establishing the reductive half-reaction as rate-limiting at physiological pH. Deuteration of the lysyl methyls results in identical kinetic isotope effects of 3.1 ± 0.2 on the kred, kcat, and kcat/Km values for the peptide, establishing C-H bond cleavage as rate-limiting with this substrate. No intermediates between oxidized and reduced flavin can be detected by stopped-flow spectroscopy, consistent with the expectation for a direct hydride transfer mechanism. The kcat/Km value for the peptide is bell-shaped, consistent with a requirement that the nitrogen at the site of oxidation be uncharged and that at least one of the other lysyl residues be charged for catalysis. The D(kcat/K m) value for the peptide is pH-independent, suggesting that the observed value is the intrinsic deuterium kinetic isotope effect for oxidation of this substrate. © 2009 American Chemical Society.

Authors
Gaweska, H; Pozzi, MH; Schmidt, DMZ; McCafferty, DG; Fitzpatrick, PF
MLA Citation
Gaweska, H, Pozzi, MH, Schmidt, DMZ, McCafferty, DG, and Fitzpatrick, PF. "Use of pH and kinetic isotope effects to establish chemistry as rate-limiting in oxidation of a peptide substrate by LSD1." Biochemistry 48.23 (2009): 5440-5445.
PMID
19408960
Source
scival
Published In
Biochemistry
Volume
48
Issue
23
Publish Date
2009
Start Page
5440
End Page
5445
DOI
10.1021/bi900499w

Probing of the cis-5-phenyl proline scaffold as a platform for the synthesis of mechanism-based inhibitors of the Staphylococcus aureus sortase SrtA isoform

cis-5-Phenyl prolinates with electrophilic substituents at the fourth position of a pyrrolidine ring were synthesized by 1,3-dipolar cycloaddition of arylimino esters with divinyl sulfone and acrylonitrile. 4-Vinylsulfonyl 5-phenyl prolinates inhibit Staphylococcus aureus sortase SrtA irreversibly by modification of the enzyme Cys184 and could be used as hits for the development of antibacterials and antivirulence agents. © 2009 Elsevier Ltd. All rights reserved.

Authors
Kudryavtsev, KV; Bentley, ML; McCafferty, DG
MLA Citation
Kudryavtsev, KV, Bentley, ML, and McCafferty, DG. "Probing of the cis-5-phenyl proline scaffold as a platform for the synthesis of mechanism-based inhibitors of the Staphylococcus aureus sortase SrtA isoform." Bioorganic and Medicinal Chemistry 17.7 (2009): 2886-2893.
PMID
19269184
Source
scival
Published In
Bioorganic & Medicinal Chemistry
Volume
17
Issue
7
Publish Date
2009
Start Page
2886
End Page
2893
DOI
10.1016/j.bmc.2009.02.008

Crystal Structure of streptococcus pyogenes Sortase A: Implications for sortase mechanism

Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. These include many proteins that play critical roles in the virulence of Gram-positive bacterial pathogens such that sortases are attractive targets for development of novel antimicrobial agents. All Gram-positive pathogens express a "housekeeping" sortase that recognizes the majority of secreted proteins containing an LPXTG wall-sorting motif and covalently attaches these to bacterial cell wall peptidoglycan. Many Gram-positive pathogens also express additional sortases that link a small number of proteins, often with variant wall-sorting motifs, to either other surface proteins or peptidoglycan. To better understand the mechanisms of catalysis and substrate recognition by the housekeeping sortase produced by the important human pathogen Streptococcus pyogenes, the crystal structure of this protein has been solved and its transpeptidase activity established in vitro. The structure reveals a novel arrangement of key catalytic residues in the active site of a sortase, the first that is consistent with kinetic analysis. The structure also provides a complete description of residue positions surrounding the active site, overcoming the limitation of localized disorder in previous structures of sortase A-type proteins. Modification of the active site Cys through oxidation to its sulfenic acid form or by an alkylating reagent supports a role for a reactive thiol/thiolate in the catalytic mechanism. These new insights into sortase structure and function could have important consequences for inhibitor design. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Race, PR; Bentley, ML; Melvin, JA; Crow, A; Hughes, RK; Smith, WD; Sessions, RB; Kehoe, MA; McCafferty, DG; Banfield, MJ
MLA Citation
Race, PR, Bentley, ML, Melvin, JA, Crow, A, Hughes, RK, Smith, WD, Sessions, RB, Kehoe, MA, McCafferty, DG, and Banfield, MJ. "Crystal Structure of streptococcus pyogenes Sortase A: Implications for sortase mechanism." Journal of Biological Chemistry 284.11 (2009): 6924-6933.
PMID
19129180
Source
scival
Published In
The Journal of biological chemistry
Volume
284
Issue
11
Publish Date
2009
Start Page
6924
End Page
6933
DOI
10.1074/jbc.M805406200

Facile synthesis of substituted trans-2-arylcyclopropylamine inhibitors of the human histone demethylase LSD1 and monoamine oxidases A and B.

A facile synthetic route to substituted trans-2-arylcyclopropylamines was developed to provide access to mechanism-based inhibitors of the human flavoenzyme oxidase lysine-specific histone demethylase LSD1 and related enzyme family members such as monoamine oxidases A and B.

Authors
Gooden, DM; Schmidt, DMZ; Pollock, JA; Kabadi, AM; McCafferty, DG
MLA Citation
Gooden, DM, Schmidt, DMZ, Pollock, JA, Kabadi, AM, and McCafferty, DG. "Facile synthesis of substituted trans-2-arylcyclopropylamine inhibitors of the human histone demethylase LSD1 and monoamine oxidases A and B." Bioorg Med Chem Lett 18.10 (May 15, 2008): 3047-3051.
PMID
18242989
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry Letters
Volume
18
Issue
10
Publish Date
2008
Start Page
3047
End Page
3051
DOI
10.1016/j.bmcl.2008.01.003

Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus

The Staphylococcus aureus transpeptidase sortase A (SrtA) is responsible for anchoring a range of virulence- and colonization-associated proteins to the cell wall. SrtA recognizes substrates that contain a C-terminal LPXTG motif. This sequence is cleaved following the threonine, and an amide bond is formed between the threonine and the pentaglycine cross-bridge of branched lipid II. Previous studies have implicated the β6/β7 loop region of SrtA in LPXTG recognition but have not systematically characterized this domain. To better understand the individual roles of the residues within this loop, we performed alanine-scanning mutagenesis. Val-168 and Leu-169 were found to be important for substrate recognition, and Glu-171 was also found to be important, consistent with its hypothesized role as a Ca2+-binding residue. Gly-167 and Asp-170 were dispensable for catalysis, as was Gln-172. The role of Arg-197 in SrtA has been the subject of much debate. To explore its role in catalysis, we used native chemical ligation to generate semi-synthetic SrtA in which we replaced Arg-197 with citrulline, a non-ionizable analog. This change resulted in a decrease of <3-fold in kcat/Km, indicating that Arg-197 utilizes a hydrogen bond, rather than an electrostatic interaction. Our results are consistent with a model for LPXTG recognition wherein the Leu-Pro sequence is recognized primarily by hydrophobic contacts with SrtA Val-168 and Leu-169, as well as a hydrogen bond from Arg-197. This model contradicts the previously proposed mechanism of binding predicted by the x-ray crystal structure of SrtA. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Bentley, ML; Lamb, EC; McCafferty, DG
MLA Citation
Bentley, ML, Lamb, EC, and McCafferty, DG. "Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus." Journal of Biological Chemistry 283.21 (2008): 14762-14771.
PMID
18375951
Source
scival
Published In
The Journal of biological chemistry
Volume
283
Issue
21
Publish Date
2008
Start Page
14762
End Page
14771
DOI
10.1074/jbc.M800974200

trans-2-Phenylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1.

The catalytic domain of the flavin-dependent human histone demethylase lysine-specific demethylase 1 (LSD1) belongs to the family of amine oxidases including polyamine oxidase and monoamine oxidase (MAO). We previously assessed monoamine oxidase inhibitors (MAOIs) for their ability to inhibit the reaction catalyzed by LSD1 [Lee, M. G., et al. (2006) Chem. Biol. 13, 563-567], demonstrating that trans-2-phenylcyclopropylamine (2-PCPA, tranylcypromine, Parnate) was the most potent with respect to LSD1. Here we show that 2-PCPA is a time-dependent, mechanism-based irreversible inhibitor of LSD1 with a KI of 242 microM and a kinact of 0.0106 s-1. 2-PCPA shows limited selectivity for human MAOs versus LSD1, with kinact/KI values only 16-fold and 2.4-fold higher for MAO B and MAO A, respectively. Profiles of LSD1 activity and inactivation by 2-PCPA as a function of pH are consistent with a mechanism of inactivation dependent upon enzyme catalysis. Mass spectrometry supports a role for FAD as the site of covalent modification by 2-PCPA. These results will provide a foundation for the design of cyclopropylamine-based inhibitors that are selective for LSD1 to probe its role in vivo.

Authors
Schmidt, DMZ; McCafferty, DG
MLA Citation
Schmidt, DMZ, and McCafferty, DG. "trans-2-Phenylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1." Biochemistry 46.14 (April 10, 2007): 4408-4416.
PMID
17367163
Source
pubmed
Published In
Biochemistry
Volume
46
Issue
14
Publish Date
2007
Start Page
4408
End Page
4416
DOI
10.1021/bi0618621

Mutational analysis of active site residues in the Staphylococcus aureus transpeptidase SrtA

In Staphylococcus aureus, virulence and colonization-associated surface proteins are covalently anchored to the cell wall by the transpeptidase Sortase A (SrtA). In order to better understand the contribution of specific active site residues to substrate recognition and catalysis, we performed mutational analysis of several key residues in the SrtA active site. Analysis of protein stability, kinetic parameters, solvent isotope effects, and pH-rate profiles for key SrtA variants are consistent with a reverse protonated Cys184-His120 catalytic dyad, and implicate a role for Arg197 in formation of an oxyanion hole to stabilize the transition state. In contrast, mutation of Asp 185 and Asp 186 produced negligible effects on catalysis, and no evidence was found to support the existence of a functional catalytic triad. Mutation of Thr180, Leu181, and Ile 182 to alanine produced modest decreases in SrtA activity and led to substrate inhibition. Thermodynamic stability measurements by SUPREX (stability of unpurified proteins from rates of H/D exchange) revealed decreases in conformational stability that correlate with the observed substrate inhibition for each variant, signifying a potential role for the conserved 180TLITC184 motif in defining the active-site architecture of SrtA. In contrast, mutation of Thr183 to alanine led to a significant 1200-fold decrease in feCat, which appears to be unrelated to conformational stability. Potential explanations for these results are discussed, and a revised model for SrtA catalysis is presented. © 2007 American Chemical Society.

Authors
Frankel, BA; Tong, Y; Bentley, ML; Fitzgerald, MC; McCafferty, DG
MLA Citation
Frankel, BA, Tong, Y, Bentley, ML, Fitzgerald, MC, and McCafferty, DG. "Mutational analysis of active site residues in the Staphylococcus aureus transpeptidase SrtA." Biochemistry 46.24 (2007): 7269-7278.
PMID
17518446
Source
scival
Published In
Biochemistry
Volume
46
Issue
24
Publish Date
2007
Start Page
7269
End Page
7278
DOI
10.1021/bi700448e

Engineering the substrate specificity of Staphylococcus aureus sortase A: The β6/β7 loop from SrtB confers npqtn recognition to SrtA

The Staphylococcus aureus transpeptidase Sortase A (SrtA) anchors virulence and colonization-associated surface proteins to the cell wall. SrtA selectively recognizes a C-terminal LPXTG motif, whereas the related transpeptidase Sortase B (SrtB) recognizes a C-terminal NPQTN motif. In both enzymes, cleavage occurs after the conserved threonine, followed by amide bond formation between threonine and the pentaglycine cross-bridge of cell wall peptidoglycan. Genetic and biochemical studies strongly suggest that SrtA and SrtB exhibit exquisite specificity for their recognition motifs. To better understand the origins of substrate specificity within these two isoforms, we used sequence and structural analysis to predict residues and domains likely to be involved in conferring substrate specificity. Mutational analyses and domain swapping experiments were conducted to test their function in substrate recognition and specificity. Marked changes in the specificity profile of SrtA were obtained by replacing the β6/β7 loop in SrtA with the corresponding domain from SrtB. The chimeric β6/β7 loop swap enzyme (SrtLS) conferred the ability to acylate NPQTN-containing substrates, with a kcat/Kmapp of 0.0062 ± 0.003 M-1 s-1. This enzyme was unable to perform the transpeptidation stage of the reaction, suggesting that additional domains are required for transpeptidation to occur. The overall catalytic specificity profile (kcat/Kmapp (NPQTN)/kcat/Kmapp (LPETG)) of SrtLS was altered 700,000-fold from SrtA. These results indicate that the β6/β7 loop is an important site for substrate recognition in sortases. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Bentley, ML; Gaweska, H; Kielec, JM; McCafferty, DG
MLA Citation
Bentley, ML, Gaweska, H, Kielec, JM, and McCafferty, DG. "Engineering the substrate specificity of Staphylococcus aureus sortase A: The β6/β7 loop from SrtB confers npqtn recognition to SrtA." Journal of Biological Chemistry 282.9 (2007): 6571-6581.
PMID
17200112
Source
scival
Published In
The Journal of biological chemistry
Volume
282
Issue
9
Publish Date
2007
Start Page
6571
End Page
6581
DOI
10.1074/jbc.M610519200

Crossing a biological velvet rope.

In contrast to our understanding of the composition of the outer membrane (OM) of Gram-negative bacteria, the biogenesis of this organelle has remained elusive. This is in part because factors involved in OM assembly have been refractive to chemical and biological analyses. A recent study shows how small molecules and chemical conditionality can be used to probe the biogenesis of the OM at the molecular level and suggests that similar techniques can be used to identify factors involved in the assembly of other organelles.

Authors
McCafferty, DG
MLA Citation
McCafferty, DG. "Crossing a biological velvet rope." ACS Chem Biol 1.6 (July 21, 2006): 339-340. (Review)
PMID
17163767
Source
pubmed
Published In
ACS Chemical Biology
Volume
1
Issue
6
Publish Date
2006
Start Page
339
End Page
340
DOI
10.1021/cb6002948

Histone H3 Lysine 4 Demethylation Is a Target of Nonselective Antidepressive Medications

Demethylation of histone H3 lysine 4 is carried out by BHC110/LSD1, an enzyme with close homology to monoamine oxidases (MAO). Monoamine oxidase A or B are frequent targets of selective and nonselective small molecular inhibitors used for treatment of depression. Here we show that in contrast to selective monoamine oxidase inhibitors such as pargyline, nonselective monoamine oxidase inhibitors potently inhibit nucleosomal demethylation of histone H3 lysine 4. Tranylcypromine (brand name Parnate) displayed the best inhibitory activity with an IC50 of less than 2 μM. Treatment of P19 embryonal carcinoma cells with tranylcypromine resulted in global increase in H3K4 methylation as well as transcriptional derepression of two BHC110 target genes, Egr1 and the pluripotent stem cell marker Oct4. These results attest to the effectiveness of tranylcypromine as a small molecular inhibitor of histone demethylation. © 2006 Elsevier Ltd. All rights reserved.

Authors
Lee, MG; Wynder, C; Schmidt, DM; McCafferty, DG; Shiekhattar, R
MLA Citation
Lee, MG, Wynder, C, Schmidt, DM, McCafferty, DG, and Shiekhattar, R. "Histone H3 Lysine 4 Demethylation Is a Target of Nonselective Antidepressive Medications." Chemistry and Biology 13.6 (2006): 563-567.
PMID
16793513
Source
scival
Published In
Chemistry & Biology
Volume
13
Issue
6
Publish Date
2006
Start Page
563
End Page
567
DOI
10.1016/j.chembiol.2006.05.004

Facile syntheses of conformationally constrained analogues of lysine and homoglutamic acid

A facile divergent synthesis of the novel amino acid trans-4-aminoethyl-l- proline and trans-4-carboxymethyl-l-proline from commercially available trans-4-hydroxy-l-proline was developed. These conformationally constrained analogues of l-lysine and l-homoglutamic acid are useful proline templated amino acids (PTAAs) with potential applications in protein engineering and de novo protein design. © 2005 Elsevier Ltd. All rights reserved.

Authors
Barkallah, S; Schneider, SL; McCafferty, DG
MLA Citation
Barkallah, S, Schneider, SL, and McCafferty, DG. "Facile syntheses of conformationally constrained analogues of lysine and homoglutamic acid." Tetrahedron Letters 46.30 (2005): 4985-4987.
Source
scival
Published In
Tetrahedron Letters
Volume
46
Issue
30
Publish Date
2005
Start Page
4985
End Page
4987
DOI
10.1016/j.tetlet.2005.05.093

Staphylococcus aureus sortase transpeptidase SrtA: Insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism

The Staphylococcus aureus transpeptidase SrtA catalyzes the covalent attachment of LPXTG-containing virulence and colonization-associated proteins to cell-wall peptidoglycan in Gram-positive bacteria. Recent structural characterizations of staphylococcal SrtA, and related transpeptidases SrtB from S. aureus and Bacillus anthracis, provide many details regarding the active site environment, yet raise questions with regard to the nature of catalysis and active site cysteine thiol activation. Here we re-evaluate the kinetic mechanism of SrtA and shed light on aspects of its catalytic mechanism. Using steady-state, pre-steady-state, bisubstrate kinetic studies, and high-resolution electrospray mass spectrometry, revised steady-state kinetic parameters and a ping-pong hydrolytic shunt kinetic mechanism were determined for recombinant SrtA. The pH dependencies of kinetic parameters kcat/Km and kcat for the substrate Abz-LPETG-Dap(Dnp)-NH2 were bell-shaped with pKa values of 6.3 ± 0.2 and 9.4 ± 0.2 for kcat and 6.2 ± 0.2 and 9.4 ± 0.2 for k cat/Km. Solvent isotope effect (SIE) measurements revealed inverse behavior, with a D2Okcat of 0.89 ± 0.01 and a D2O(kcat/Km) of 0.57 ± 0.03 reflecting an equilibrium SIE. In addition, SIE measurements strongly implicated Cys184 participation in the isotope-sensitive rate-determining chemical step when considered in conjunction with an inverse linear proton inventory for kcat. Last, the pH dependence of SrtA inactivation by iodoacetamide revealed a single ionization for inactivation. These studies collectively provide compelling evidence for a reverse protonation mechanism where a small fraction (ca. 0.06%) of SrtA is competent for catalysis at physiological pH, yet is highly active with an estimated kcat/Km of >10 5 M-1 s-1. © 2005 American Chemical Society.

Authors
Frankel, BA; Kruger, RG; Robinson, DE; Kelleher, NL; McCafferty, DG
MLA Citation
Frankel, BA, Kruger, RG, Robinson, DE, Kelleher, NL, and McCafferty, DG. "Staphylococcus aureus sortase transpeptidase SrtA: Insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism." Biochemistry 44.33 (2005): 11188-11200.
PMID
16101303
Source
scival
Published In
Biochemistry
Volume
44
Issue
33
Publish Date
2005
Start Page
11188
End Page
11200
DOI
10.1021/bi050141j

Solvent Dependence of Intramolecular Electron Transfer in a Helical Oligoproline Assembly

The helical oligoproline assembly CH 3-CO-Pro-Pro-Pro-Pra(Ptzpn)-Pro-Pro-Pra(RuIIb 2m)2+-Pro-Pro-Pra(Anq)-Pro-Pro-Pro-NH2 having a spatially ordered array of functional sites protruding from the proline backbone, has been prepared. The 13-residue assembly formed a linear array containing a phenothiazine electron donor, a tris(bipyridine)ruthenium(II) chromophore, and an anthraquinone electron acceptor with the proline II secondary structure as shown by circular dichroism measurements. Following RuII → b2m metal-to-ligand charge-transfer (MLCT) excitation at 457 nm, electron-transfer quenching occurs, ultimately to give a redox-separated (RS) state containing a phenothiazine (PTZ) radical cation at the Pra(Ptzpn) site and an anthraquinone (ANQ) radical anion at the Pra(Anq) site. The redox-separated state was formed with 33-96% efficiency depending on the solvent, and the transient stored energy varied from -1.46 to -1.71 eV at 22 ± 2 °C. The dominant quenching mechanism is PTZ reductive quenching of the initial RuIII(b2m.-) MLCT excited state which is followed by m.- → ANQ electron transfer to give the RS state. Back electron transfer is highly exergonic and occurs in the inverted region. The rate constant for back electron transfer is solvent dependent and varies from 5.2 × 106 to 7.7 × 10 6 s-1 at 22 ± 2 °C. It is concluded that back electron transfer occurs by direct ANQ.- → PTZ.+ electron transfer. Based on independently evaluated kinetic parameters, the electron-transfer matrix element is HDA ≈ 0.13 cm-1.

Authors
Striplin, DR; Reece, SY; McCafferty, DG; Wall, CG; Friesen, DA; Erickson, BW; Meyer, TJ
MLA Citation
Striplin, DR, Reece, SY, McCafferty, DG, Wall, CG, Friesen, DA, Erickson, BW, and Meyer, TJ. "Solvent Dependence of Intramolecular Electron Transfer in a Helical Oligoproline Assembly." Journal of the American Chemical Society 126.16 (2004): 5282-5291.
PMID
15099113
Source
scival
Published In
Journal of the American Chemical Society
Volume
126
Issue
16
Publish Date
2004
Start Page
5282
End Page
5291
DOI
10.1021/ja0304289

Inhibition of the Staphylococcus aureus sortase transpeptidase SrtA by phosphinic peptidomimetics

During pathogenesis, Gram-positive bacteria utilize surface protein virulence factors such as the MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) to aid the initiation and propagation of infection through adherence to host endothelial tissue and immune system evasion. These virulence-associated proteins generally contain a C-terminal LPXTG motif that becomes covalently anchored to the peptidoglycan biosynthesis intermediate lipid II. In Staphylococcus aureus, deletion of the sortase isoform SrtA results in marked reduction in virulence and infection potential, making it an important antivirulence target. Here we describe the chemical synthesis and kinetic characterization of a nonhydrolyzable phosphinic peptidomimetic inhibitor of SrtA derived from the LPXTG substrate sequence. © 2004 Elsevier Ltd. All rights reserved.

Authors
Kruger, RG; Barkallah, S; Frankel, BA; McCafferty, DG
MLA Citation
Kruger, RG, Barkallah, S, Frankel, BA, and McCafferty, DG. "Inhibition of the Staphylococcus aureus sortase transpeptidase SrtA by phosphinic peptidomimetics." Bioorganic and Medicinal Chemistry 12.13 (2004): 3723-3729.
PMID
15186858
Source
scival
Published In
Bioorganic and Medicinal Chemistry
Volume
12
Issue
13
Publish Date
2004
Start Page
3723
End Page
3729
DOI
10.1016/j.bmc.2004.03.066

A suppression strategy for antibiotic discovery

High-throughput phenotype screening and target identification have been combined in an effort to isolate antimicrobial, small-molecule therapeutics [1]. This approach, developed by Brown and colleagues and reported in this issue, is a major technological advance for antimicrobial drug discovery.

Authors
Gaweska, H; Kielec, J; McCafferty, D
MLA Citation
Gaweska, H, Kielec, J, and McCafferty, D. "A suppression strategy for antibiotic discovery." Chemistry and Biology 11.10 (2004): 1330-1332.
PMID
15489159
Source
scival
Published In
Chemistry & Biology
Volume
11
Issue
10
Publish Date
2004
Start Page
1330
End Page
1332
DOI
10.1016/j.chembiol.2004.09.007

Profiling natural product biosynthesis

Natural products are a rich source of therapeutics; however, artificially reengineering the biosynthetic pathways that generate these compounds could potentially generate "designer" drugs. Last month in Chemistry & Biology, Burkart and coworkers reported their technique to track and better understand the components of these pathways [1].

Authors
Frankel, BA; McCafferty, DG
MLA Citation
Frankel, BA, and McCafferty, DG. "Profiling natural product biosynthesis." Chemistry and Biology 11.3 (2004): 290-291.
PMID
15123256
Source
scival
Published In
Chemistry & Biology
Volume
11
Issue
3
Publish Date
2004
Start Page
290
End Page
291
DOI
10.1016/j.chembiol.2004.03.007

Vinyl Sulfones: Inhibitors of SrtA, a Transpeptidase Required for Cell Wall Protein Anchoring and Virulence in Staphylococcus aureus

Several small molecule vinyl sulfones were found to exhibit irreversible time-dependent inhibition of the Staphylococcus aureus sortase SrtA in vitro. A representative of these compounds was shown to impair the ability of S. aureus bacteria to bind fibronectin-coated surfaces through in vivo inhibition of SrtA-mediated linkage of fibronectin to the cell surface. These data highlight the potential use of small molecule vinyl sulfones as chemotherapeutics to prevent adhesion to and colonization of host tissues during S. aureus infection. Copyright © 2004 American Chemical Society.

Authors
Frankel, BA; Bentley, M; Kruger, RG; McCafferty, DG
MLA Citation
Frankel, BA, Bentley, M, Kruger, RG, and McCafferty, DG. "Vinyl Sulfones: Inhibitors of SrtA, a Transpeptidase Required for Cell Wall Protein Anchoring and Virulence in Staphylococcus aureus." Journal of the American Chemical Society 126.11 (2004): 3404-3405.
PMID
15025450
Source
scival
Published In
Journal of the American Chemical Society
Volume
126
Issue
11
Publish Date
2004
Start Page
3404
End Page
3405
DOI
10.1021/ja0390294

Analysis of the Substrate Specificity of the Staphylococcus aureus Sortase Transpeptidase SrtA

The Staphylococcus aureus sortase transpeptidase SrtA isoform is responsible for the covalent attachment of virulence and colonization-associated proteins to the bacterial peptidoglycan. SrtA utilizes two substrates, undecaprenol-pyrophosphoryl-MurNAc(GlcNAc)-Ala-D-isoGlu-Lys(Ε-Gly 5)-D-Ala-D-Ala (branched Lipid II) and secreted proteins containing a highly conserved C-terminal LPXTG sequence. SrtA simultaneously cleaves the Thr-Gly bond of the LPXTG-containing protein and forms a new amide bond with the nucleophilic amino group of the Gly5 portion of branched Lipid II, anchoring the protein to this key intermediate that is subsequently polymerized into peptidoglycan. Here we describe the development of a general in vitro method for elucidating the substrate specificity of sortase enzymes. In addition, using immunofluorescence, cell adhesion assays, and transmission electron microscopy, we establish links between in vitro substrate specificity and in vivo function of the S. aureus sortase isoforms. Results from these studies provide strong supporting evidence of a primary role of the SrtA isoform in S. aureus adhesion and host colonization, illustrate a lack of specificity cross talk between SrtA and SrtB isoforms, and highlight the potential of SrtA as a target for the development of antivirulence chemotherapeutics against Gram-positive bacterial pathogens.

Authors
Kruger, RG; Otvos, B; Frankel, BA; Bentley, M; Dostal, P; McCafferty, DG
MLA Citation
Kruger, RG, Otvos, B, Frankel, BA, Bentley, M, Dostal, P, and McCafferty, DG. "Analysis of the Substrate Specificity of the Staphylococcus aureus Sortase Transpeptidase SrtA." Biochemistry 43.6 (2004): 1541-1551.
PMID
14769030
Source
scival
Published In
Biochemistry
Volume
43
Issue
6
Publish Date
2004
Start Page
1541
End Page
1551
DOI
10.1021/bi035920j

Development of a high-performance liquid chromatography assay and revision of kinetic parameters for the Staphylococcus aureus sortase transpeptidase SrtA

The SrtA isoform of the Staphylococcus aureus sortase transpeptidase is responsible for the covalent attachment of virulence- and colonization- associated proteins to the bacterial peptidoglycan. Sortase utilizes two substrates, undecaprenol-pyrophosphoryl-MurNAc(GlcNAc)-Ala-D-isoGlu-Lys(ε- Gly5)-D-Ala-D-Ala (branched Lipid II) and secreted proteins containing a highly conserved LPXTG sequence near their C termini. SrtA simultaneously cleaves the Thr-Gly bond of the LPXTG-containing protein and forms a new amide bond with the nucleophilic amino group of the Gly5 portion of branched Lipid II, anchoring the protein to this key intermediate that is subsequently polymerized into peptidoglycan. Here we show that reported fluorescence quenching activity assays for SrtA are subject to marked fluorescence inner filter effect quenching, resulting in prematurely hyperbolic velocity versus substrate profiles and underestimates of the true kinetic parameters kcat and Km. We therefore devised a discontinuous high-performance liquid chromatography (HPLC)-based assay to monitor the SrtA reaction employing the same substrates used in the fluorescence quenching assay: Gly5 and Abz-LPETG-Dap(Dnp)-NH 2. Fluorescence or UV detection using these substrates facilitates separate analysis of both the acylation and the transpeptidation steps of the reaction. Because HPLC was performed using fast-flow analytical columns (<8min/run), high-throughput applications of this assay for analysis of SrtA substrate specificity, kinetic mechanism, and inhibition are now feasible. Kinetic analysis using the HPLC assay revealed that the kinetic parameters for SrtA with Abz-LPETG-Dap(Dnp)-NH2 are 5.5mM for Km and 0.27s-1 for kcat. The Km for Gly5 was determined to be 140μM. These values represent a 300-fold increase in Km for the LPXTG substrate and a 12,000-fold increase in k cat over literature-reported values, suggesting that SrtA is more a robust enzyme than previous analyses indicated. © 2003 Elsevier Inc. All rights reserved.

Authors
Kruger, RG; Dostal, P; McCafferty, DG
MLA Citation
Kruger, RG, Dostal, P, and McCafferty, DG. "Development of a high-performance liquid chromatography assay and revision of kinetic parameters for the Staphylococcus aureus sortase transpeptidase SrtA." Analytical Biochemistry 326.1 (2004): 42-48.
PMID
14769334
Source
scival
Published In
Analytical Biochemistry
Volume
326
Issue
1
Publish Date
2004
Start Page
42
End Page
48
DOI
10.1016/j.ab.2003.10.023

Techniques Used to Study Transcription on Chromatin Templates

Authors
Loyola, A; He, S; Oh, S; McCafferty, DG; Reinberg, D
MLA Citation
Loyola, A, He, S, Oh, S, McCafferty, DG, and Reinberg, D. "Techniques Used to Study Transcription on Chromatin Templates." Methods in Enzymology 377 (2004): 474-499.
PMID
14979046
Source
scival
Published In
Methods in Enzymology
Volume
377
Publish Date
2004
Start Page
474
End Page
499
DOI
10.1016/S0076-6879(03)77031-1

Facile synthesis of site-specifically acetylated and methylated histone proteins: Reagents for evaluation of the histone code hypothesis

The functional capacity of genetically encoded histone proteins can be powerfully expanded by posttranslational modification. A growing body of biochemical and genetic evidence clearly links the unique combinatorial patterning of side chain acetylation, methylation, and phosphorylation mainly within the highly conserved N termini of histones H2A, H2B, H3, and H4 with the regulation of gene expression and chromatin assembly and remodeling, in effect constituting a "histone code" for epigenetic signaling. Deconvoluting this code has proved challenging given the inherent posttranslational heterogeneity of histone proteins isolated from biological sources. Here we describe the application of native chemical ligation to the preparation of full-length histone proteins containing site-specific acetylation and methylation modifications. Peptide thioesters corresponding to histone N termini were prepared by solid phase peptide synthesis using an acid labile Boc/HF assembly strategy, then subsequently ligated to recombinantly produced histone C-terminal globular domains containing an engineered N-terminal cysteine residue. The ligation site is then rendered traceless by hydrogenolytic desulfurization, generating a native histone protein sequence. Synthetic histones generated by this method are fully functional, as evidenced by their self-assembly into a higher order H3/H4 heterotetramer, their deposition into nucleosomes by human ISWI-containing (Imitation of Switch) factor RSF (Remodeling and Spacing factor), and by enzymatic modification by human Sirt1 deacetylase and G9a methyltransferase. Site-specifically modified histone proteins generated by this method will prove invaluable as novel reagents for the evaluation of the histone code hypothesis and analysis of epigenetic signaling mechanisms.

Authors
He, S; Bauman, D; Davis, JS; Loyola, A; Nishioka, K; Gronlund, JL; Reinberg, D; Meng, F; Kelleher, N; McCafferty, DG
MLA Citation
He, S, Bauman, D, Davis, JS, Loyola, A, Nishioka, K, Gronlund, JL, Reinberg, D, Meng, F, Kelleher, N, and McCafferty, DG. "Facile synthesis of site-specifically acetylated and methylated histone proteins: Reagents for evaluation of the histone code hypothesis." Proceedings of the National Academy of Sciences of the United States of America 100.21 (2003): 12033-12038.
PMID
14530408
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
100
Issue
21
Publish Date
2003
Start Page
12033
End Page
12038
DOI
10.1073/pnas.2035256100

Phosphorus-based SAHA analogues as histone deacetylase inhibitors

(Matrix presented) Three analogues of suberoyl anilide hydroxamic acid (SAHA) with phosphorus metal-chelating functionalities were synthesized as inhibitors of histone deacetylases (HDACs). The compounds showed weak activity for HeLa nuclear extracts (IC50 = 0.57-6.1 mM), HDAC8 (IC 50 = 0.28-0.41 mM), and histone-deacetylase-like protein (HDLP, IC50 = 0.33-1.9 mM), suggesting that the transition state of HDAC is not analogous to zinc proteases. Antiproliferative activity against A2780 cancer cells (IC50 = 0.11-0.12 mM), comparable to SAHA (0.15 mM), was observed.

Authors
Kapustin, GV; Fejér, G; Gronlund, JL; McCafferty, DG; Seto, E; Etzkorn, FA
MLA Citation
Kapustin, GV, Fejér, G, Gronlund, JL, McCafferty, DG, Seto, E, and Etzkorn, FA. "Phosphorus-based SAHA analogues as histone deacetylase inhibitors." Organic Letters 5.17 (2003): 3053-3056.
PMID
12916979
Source
scival
Published In
Organic Letters
Volume
5
Issue
17
Publish Date
2003
Start Page
3053
End Page
3056
DOI
10.1021/ol035056n

An economical and preparative orthogonal solid phase synthesis of fluorescein and rhodamine derivatized peptides: FRET substrates for the Staphylococcus aureus sortase SrtA transpeptidase reaction.

An economical and preparative-scale orthogonal solid-phase method of incorporating carboxyrhodamine and carboxyfluorescein fluorescence resonance energy transfer (FRET) probes site-specifically into synthetic peptide substrates for the S. aureus Sortase transpeptidase SrtA has been developed.

Authors
Kruger, RG; Dostal, P; McCafferty, DG
MLA Citation
Kruger, RG, Dostal, P, and McCafferty, DG. "An economical and preparative orthogonal solid phase synthesis of fluorescein and rhodamine derivatized peptides: FRET substrates for the Staphylococcus aureus sortase SrtA transpeptidase reaction." Chemical communications (Cambridge, England) 18 (2002): 2092-2093.
Source
scival
Published In
Chemical communications (Cambridge, England)
Issue
18
Publish Date
2002
Start Page
2092
End Page
2093

Light-induced helix formation

This study shows that incorporation of [Rub2m-OH]2+ at the N-terminus of the Fs peptide enhances its stability by∼0.15 kcal/mol through the mechanism of dipole-dipole coupling at the excited state, suggesting that photoinduced charge generation at a well-controlled and specific location provides a convenient means to trigger helix-coil transition on nanosecond or even faster time scales. Copyright © 2002 American Chemical Society.

Authors
Huang, C-Y; He, S; DeGrado, WF; McCafferty, DG; Gai, F
MLA Citation
Huang, C-Y, He, S, DeGrado, WF, McCafferty, DG, and Gai, F. "Light-induced helix formation." Journal of the American Chemical Society 124.43 (2002): 12674-12675.
Source
scival
Published In
Journal of the American Chemical Society
Volume
124
Issue
43
Publish Date
2002
Start Page
12674
End Page
12675
DOI
10.1021/ja028084u

Functional Analysis of the Lipoglycodepsipeptide Antibiotic Ramoplanin

The peptide antibiotic ramoplanin is highly effective against several drug-resistant gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), two important opportunistic human pathogens. Ramoplanin inhibits bacterial peptidoglycan (PG) biosynthesis by binding to Lipid intermediates I and II at a location different than the N-acyl-D-Ala-D-Ala dipeptide site targeted by vancomycin. Lipid I/II capture physically occludes these substrates from proper utilization by the late-stage PG biosynthesis enzymes MurG and the transglycosylases. Key structural features of ramoplanin responsible for antibiotic activity and PG molecular recognition have been discovered by antibiotic semisynthetic modification in conjunction with NMR analyses. These results help define a minimalist ramoplanin pharmacophore and introduce the possibility of generating ramoplanin-derived peptide or peptidomimetic antibiotics for use against VRE, MRSA, and related pathogens.

Authors
Cudic, P; Behenna, DC; Kranz, JK; Kruger, RG; Wand, AJ; Veklich, YI; Weisel, JW; McCafferty, DG
MLA Citation
Cudic, P, Behenna, DC, Kranz, JK, Kruger, RG, Wand, AJ, Veklich, YI, Weisel, JW, and McCafferty, DG. "Functional Analysis of the Lipoglycodepsipeptide Antibiotic Ramoplanin." Chemistry and Biology 9.8 (2002): 897-906.
PMID
12204689
Source
scival
Published In
Chemistry & Biology
Volume
9
Issue
8
Publish Date
2002
Start Page
897
End Page
906
DOI
10.1016/S1074-5521(02)00191-6

Chemistry and biology of the ramoplanin family of peptide antibiotics

The peptide antibiotic ramoplanin factor A2 is a promising clinical candidate for treatment of Gram-positive bacterial infections that are resistant to antibiotics such as glycopeptides, macrolides, and penicillins. Since its discovery in 1984, no clinical or laboratory-generated resistance to this antibiotic has been reported. The mechanism of action of ramoplanin involves sequestration of peptidoglycan biosynthesis Lipid intermediates, thus physically occluding these substrates from proper utilization by the late-stage peptidoglycan biosynthesis enzymes MurG and the transglycosylases (TGases). Ramoplanin is structurally related to two cell wall active lipodepsipeptide antibiotics, janiemycin, and enduracidin, and is functionally related to members of the lantibiotic class of antimicrobial peptides (mersacidin, actagardine, nisin, and epidermin) and glycopeptide antibiotics (vancomycin and teicoplanin). Peptidomimetic chemotherapeutics derived from the ramoplanin sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and related pathogens. Here we review the chemistry and biology of the ramoplanins including its discovery, structure elucidation, biosynthesis, antimicrobial activity, mechanism of action, and total synthesis. © 2002 Wiley Periodicals, Inc.

Authors
McCafferty, DG; Cudic, P; Frankel, BA; Barkallah, S; Kruger, RG; Li, W
MLA Citation
McCafferty, DG, Cudic, P, Frankel, BA, Barkallah, S, Kruger, RG, and Li, W. "Chemistry and biology of the ramoplanin family of peptide antibiotics." Biopolymers - Peptide Science Section 66.4 (2002): 261-284.
PMID
12491539
Source
scival
Published In
Biopolymers - Peptide Science Section
Volume
66
Issue
4
Publish Date
2002
Start Page
261
End Page
284
DOI
10.1002/bip.10296

Complexation of peptidoglycan intermediates by the lipoglycodepsipeptide antibiotic ramoplanin: Minimal structural requirements for intermolecular complexation and fibril formation

The peptide antibiotic ramoplanin inhibits bacterial peptidoglycan (PG) biosynthesis by interrupting late-stage membrane-associated glycosyltransferase reactions catalyzed by the transglycosylase and MurG enzymes. The mechanism of ramoplanin involves sequestration of lipid-anchored PG biosynthesis intermediates, physically occluding these substrates from proper utilization by these enzymes. In this report, we describe the first molecular-level details of the interaction of ramoplanin with PG biosynthesis intermediates. NMR analysis in conjunction with chemical dissection of the PG monomer revealed that the ramoplanin octapeptide D-Hpg-D-Orn-D-alloThr-Hpg-D-Hpg-alloThr-Phe-D-Orn recognizes MurNAc-Ala-γ-D-Glu pyrophosphate, the minimum component of PG capable of high-affinity complexation and fibril formation. Ramoplanin therefore recognizes a PG binding locus different from the N-acyl-D-Ala-D-Ala moiety targeted by vancomycin. Because ramoplanin is structurally less complex than glycopeptide antibiotics such as vancomycin, peptidomimetic chemotherapeutics derived from this recognition sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, and related pathogens.

Authors
Cudic, P; Kranz, JK; Behenna, DC; Kruger, RG; Tadesse, H; Wand, AJ; Veklich, YI; Weisel, JW; McCafferty, DG
MLA Citation
Cudic, P, Kranz, JK, Behenna, DC, Kruger, RG, Tadesse, H, Wand, AJ, Veklich, YI, Weisel, JW, and McCafferty, DG. "Complexation of peptidoglycan intermediates by the lipoglycodepsipeptide antibiotic ramoplanin: Minimal structural requirements for intermolecular complexation and fibril formation." Proceedings of the National Academy of Sciences of the United States of America 99.11 (2002): 7384-7389.
PMID
12032292
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
99
Issue
11
Publish Date
2002
Start Page
7384
End Page
7389
DOI
10.1073/pnas.102192099

An economical and preparative orthogonal solid phase synthesis of fluorescein and rhodamine derivatized peptides: FRET substrates for the Staphylococcus aureus sortase SrtA transpeptidase reaction

An economical and preparative-scale orthogonal solid-phase method of incorporating carboxyrhodamine and carboxy-fluorescein fluorescence resonance energy transfer (FRET) probes site-specifically into synthetic peptide substrates for the S. aureus Sortase transpeptidase SrtA has been developed.

Authors
Kruger, RG; Dostal, P; McCafferty, DG
MLA Citation
Kruger, RG, Dostal, P, and McCafferty, DG. "An economical and preparative orthogonal solid phase synthesis of fluorescein and rhodamine derivatized peptides: FRET substrates for the Staphylococcus aureus sortase SrtA transpeptidase reaction." Chemical Communications 18 (2002): 2092-2093.
Source
scival
Published In
Chemical Communications
Issue
18
Publish Date
2002
Start Page
2092
End Page
2093

Synthesis of P1-Citronellyl-P2-α-D-pyranosyl pyrophosphates as potential substrates for the E. coli undecaprenyl-pyrophosphoryl-N-acetylglucoseaminyl transferase MurG

P1-Citronellyl-P2-α-D-pyranosyl pyrophosphates containing α-D-N-acetylglucoseaminyl, α-D-glucosyl, and α-D-N-acetylmuramyl carbohydrates were synthesized and used in substrate specificity studies of the Escherichia coli MurG enzyme. Oxalyl chloride activation of citronellyl phosphate for coupling to α-D-pyranose-1-phosphates resulted in markedly improved yields over traditional Khorana-Moffatt and diphenyl chlorophosphate activation strategies. © 2001 Elsevier Science Ltd. All rights reserved.

Authors
Cudic, P; Behenna, DC; Yu, MK; Kruger, RG; Szewczuk, LM; McCafferty, DG
MLA Citation
Cudic, P, Behenna, DC, Yu, MK, Kruger, RG, Szewczuk, LM, and McCafferty, DG. "Synthesis of P1-Citronellyl-P2-α-D-pyranosyl pyrophosphates as potential substrates for the E. coli undecaprenyl-pyrophosphoryl-N-acetylglucoseaminyl transferase MurG." Bioorganic and Medicinal Chemistry Letters 11.24 (2001): 3107-3110.
PMID
11720853
Source
scival
Published In
Bioorganic & Medicinal Chemistry Letters
Volume
11
Issue
24
Publish Date
2001
Start Page
3107
End Page
3110
DOI
10.1016/S0960-894X(01)00653-9

4'-Aminomethyl-2,2'-bipyridyl-4-carboxylic acid (Abc) and related derivatives: Novel bipyridine amino acids for the solid-phase incorporation of a metal coordination site within a peptide backbone

The novel bipyridyl amino acid, 4'-aminomethyl-2,2'-bipyridyl-4- carboxylic acid (Abc), and related Boc- and Fmoc-protected derivatives were synthesized to provide high-affinity bidentate metal-binding amino acid modules for the solid-phase peptide synthesis (SPPS) of metallopeptides. Since the bipyridyl group of Abc is inserted into the peptide mainchain and not in the sidechain, its presence in a peptide should impart distinct conformational constraints to the backbone geometry, influencing local secondary structure. To demonstrate its amenability for SPPS and its capacity for metal complexation, Abc was incorporated into the hexapeptide Ac-Ala-Abc- Ahx-Ahx-Abc-Gly-NH2 (peptide Aha; where Ahx=aminohexanoic acid) and subsequently used as a tetradentate ligand to octahedrally coordinate and asymmetrically encapsulate a ruthenium(II) ion, creating a novel peptide- caged redox-active metal complex. (C) 2000 Elsevier Science Ltd.

Authors
Bishop, BM; McCafferty, DG; Erickson, BW
MLA Citation
Bishop, BM, McCafferty, DG, and Erickson, BW. "4'-Aminomethyl-2,2'-bipyridyl-4-carboxylic acid (Abc) and related derivatives: Novel bipyridine amino acids for the solid-phase incorporation of a metal coordination site within a peptide backbone." Tetrahedron 56.27 (2000): 4629-4638.
Source
scival
Published In
Tetrahedron
Volume
56
Issue
27
Publish Date
2000
Start Page
4629
End Page
4638

Synergy and duality in peptide antibiotic mechanisms

The molecular mechanisms by which peptide antibiotics disrupt bacterial DNA synthesis, protein biosynthesis, cell wall biosynthesis, and membrane integrity are diverse, yet historically have been understood to follow a theme of one antibiotic, one inhibitory mechanism. In the past year, mechanistic and structural studies have shown a rich diversity in peptide antibiotic mechanism. Novel secondary targeting mechanisms for peptide antibiotics have recently been discovered, and the mechanisms of peptide antibiotics involved in synergistic relationships with antibiotics and proteins have been more clearly defined. In apparent response to selective pressures, antibiotic-producing organisms have elegantly integrated multiple functions and cooperative interactions into peptide antibiotic design for the purpose of improving antimicrobial success.

Authors
McCafferty, DG; Cudic, P; Yu, MK; Behenna, DC; Kruger, R
MLA Citation
McCafferty, DG, Cudic, P, Yu, MK, Behenna, DC, and Kruger, R. "Synergy and duality in peptide antibiotic mechanisms." Current Opinion in Chemical Biology 3.6 (1999): 672-680.
PMID
10600730
Source
scival
Published In
Current Opinion in Chemical Biology
Volume
3
Issue
6
Publish Date
1999
Start Page
672
End Page
680
DOI
10.1016/S1367-5931(99)00025-3

Homologs of the vancomycin resistance D-Ala-D-Ala dipeptidase VanX in Streptomyces toyocaensis, Escherichia coli and Synechocystis: Attributes of catalytic efficiency, stereoselectivity and regulation with implications for function

Background: Vancomycin-resistant enterococci are pathogenic bacteria that have altered cell-wall peptidoglycan termini (D-alanyl-D-lactate [D-Ala-D-lactate] instead of D-alanyl-D-alanine [D-Ala-D-Ala]), which results in a 1000-fold decreased affinity for binding vancomycin. The metallodipeptidase VanX (EntVanX) is a key enzyme in antibiotic resistance as it reduces the cellular pool of the D-Ala-D-Ala dipeptide. Results: A bacterial genome search revealed vanX homologs in Streptomyces toyocaensis (StoVanX), Escherichia coli (EcoVanX), and Synechocystis sp. strain PCC6803 (SynVanX). Here, the D,D-dipeptidase catalytic activity of all three VanX homologs is validated, and the catalytic efficiencies and diastereoselectivity ratios for dipeptide cleavage are reported. The ecovanX gene is shown to have an RpoS (σ(s))-dependent promoter typical of genes turned on in stationary phase. Expression of ecovanX and an associated cluster of dipeptide permease genes permitted growth of E. coli using D-Ala-D-Ala as the sole carbon source. Conclusions: The key residues of the EntVanX active site are strongly conserved in the VanX homologs, suggesting their active-site topologies are similar. StoVanX is a highly efficient D-Ala-D-Ala dipeptidase; its gene is located in a vanHAX operon, consistent with a vancomycin-immunity function. StoVanX is a potential source for the VanX found in gram-positive enterococci. The catalytic efficiencies of D-Ala-D-Ala hydrolysis for EcoVanX and SynVanX are 25-fold lower than for EntVanX, suggesting they have a role in cell-wall turnover. Clustered with the ecovanX gene is a putative dipeptide permease system that imports D-Ala-D-Ala into the cell. The combined action of EcoVanX and the permease could permit the use of D-Ala-D-Ala as a bacterial energy source under starvation conditions.

Authors
Lessard, IAD; Pratt, SD; McCafferty, DG; Bussiere, DE; Hutchins, C; Wanner, BL; Katz, L; Walsh, CT
MLA Citation
Lessard, IAD, Pratt, SD, McCafferty, DG, Bussiere, DE, Hutchins, C, Wanner, BL, Katz, L, and Walsh, CT. "Homologs of the vancomycin resistance D-Ala-D-Ala dipeptidase VanX in Streptomyces toyocaensis, Escherichia coli and Synechocystis: Attributes of catalytic efficiency, stereoselectivity and regulation with implications for function." Chemistry and Biology 5.9 (1998): 489-504.
PMID
9751644
Source
scival
Published In
Chemistry & Biology
Volume
5
Issue
9
Publish Date
1998
Start Page
489
End Page
504

Mutational analysis of potential zinc-binding residues in the active site of the enterococcal D-Ala-D-Ala dipeptidase VanX

VanX, one of the five proteins required for the vancomycin-resistant phenotype in clinically pathogenic Enterococci, is a zinc-containing D-Ala- D-Ala dipeptidase. To identify potentia zinc ligands and begin defining the active site residues, we have mutated the 2 cysteine, 5 histidine, and 4 of the 28 aspartate and glutamate residues in the 202 residue VanX protein. Of 10 mutations, 3 cause inactivation and greater than 90% loss of zinc in purified enzyme samples, implicating His116, Asp123, and His184 as zinc- coordinating residues. Homology searches using the 10 amino acid sequence SxHxxGxAxD, in which histidine and aspartate residues are putative zinc ligands, identified the metal coordinating ligands in the N-terminal domain of the murine Sonic hedgehog protein, which also exhibits an architecture for metal coordination identical to that observed in thermolysin from Bacillus thermoproteolyticus. Furthermore, this 10 amino acid consensus sequence is found in the Streptomyces albus G zinc-dependent N-acyl-D-Ala-D-Ala carboxypeptidase, an enzyme catalyzing essentially the same D-Ala-D-Ala dipeptide bond cleavage as VanX, suggesting equivalent mechanisms and zinc catalytic site architectures. VanX residue Glu181 is analogous to the Glu143 catalytic base in B. thermoproteolyticus thermolysin, and the E181A VanX mutant has no detectable dipeptidase activity, yet maintains near- stoichiometric zinc content, a result consistent with the participation of the residue as a catalytic base.

Authors
McCafferty, DG; Lessard, IAD; Walsh, CT
MLA Citation
McCafferty, DG, Lessard, IAD, and Walsh, CT. "Mutational analysis of potential zinc-binding residues in the active site of the enterococcal D-Ala-D-Ala dipeptidase VanX." Biochemistry 36.34 (1997): 10498-10505.
PMID
9265630
Source
scival
Published In
Biochemistry
Volume
36
Issue
34
Publish Date
1997
Start Page
10498
End Page
10505
DOI
10.1021/bi970543u

Photochemical energy conversion in a helical oligoproline assembly

A general method is described for constructing a helical oligoproline assembly having a spatially ordered array of functional sites protruding from a proline-II helix. Three different redox-active carboxylic acids were coupled to the side chain of cis-4-amino-L-proline. These redox modules were incorporated through solid-phase peptide synthesis into a 13-residue helical oligoproline assembly bearing in linear array a phenothiazine electron donor, a tris(bipyridine)ruthenium(II) chromophore, and an anthraquinone electron acceptor. Upon transient 460-nm irradiation in acetonitrile, this peptide triad formed with 53% efficiency an excited state containing a phenothiazine radical cation and an anthraquinone radical anion. This light-induced redox- separated state had a lifetime of 175 ns and stored 1.65 eV of energy.

Authors
McCafferty, DG; Friesen, DA; Danielson, E; Wall, CG; Saderholm, MJ; Erickson, BW; Meyer, TJ
MLA Citation
McCafferty, DG, Friesen, DA, Danielson, E, Wall, CG, Saderholm, MJ, Erickson, BW, and Meyer, TJ. "Photochemical energy conversion in a helical oligoproline assembly." Proceedings of the National Academy of Sciences of the United States of America 93.16 (1996): 8200-8204.
PMID
8710847
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
93
Issue
16
Publish Date
1996
Start Page
8200
End Page
8204
DOI
10.1073/pnas.93.16.8200

Engineering of a 129-residue tripod protein by chemoselective ligation of proline-II helices

A 129-residue tripod protein was designed, synthesized, and biophysically characterized. This receptor-adhesive modular protein contained three 30-residue proline-II helices linked to a 9-residue proline-II helix through thioether bonds. Coupling of 6-maleimidohexanoic acid succinimido ester to cis-Nα-Boc-4-amino-L-proline furnished in 77% yield the maleimido acid cis-N-Boc-4-(6-maleimidohexanamido)-L-proline (Boc-Prm), which was used in the solid-phase synthesis of the linker peptide CH3-CO-Pro3-Prm3-Pro3-***NH2. The leg peptide, the 40-residue thiol Gly-Arg-Gly-Asp-Ser-Pro-Gly-Tyr-Gly-Pro30-Cys-NH2, was also made by solid-phase synthesis. The tripod protein was prepared by Michael addition of the thiol groups of three leg peptides to the three maleimide groups of the linker peptide. By 13C NMR spectrometry, the linker peptide was a proline-II helix, as indicated by the presence of only trans Pro-Pro resonances for its β and γ carbons. By circular dichroic spectroscopy, the model peptide CH3-CO-Pro9-NH2, the linker peptide, the leg peptide, and the tripod protein each contained substantial proline-II helix, as indicated by a strong negative band at 205 nm and a weak positive band at 226 nm. Since the Pro30 proline-II helix of each leg is about 93 Å long, two Arg-Gly-Asp sites on different legs of the tripod protein could be as much as ≈250 Å apart. © 1995 Elsevier Science Ltd.

Authors
McCafferty, DG; Slate, CA; Nakhle, BM; Jr, HDG; Austell, TL; Vachet, RW; Mullis, BH; Erickson, BW
MLA Citation
McCafferty, DG, Slate, CA, Nakhle, BM, Jr, HDG, Austell, TL, Vachet, RW, Mullis, BH, and Erickson, BW. "Engineering of a 129-residue tripod protein by chemoselective ligation of proline-II helices." Tetrahedron 51.36 (1995): 9859-9872.
Source
scival
Published In
Tetrahedron
Volume
51
Issue
36
Publish Date
1995
Start Page
9859
End Page
9872
DOI
10.1016/0040-4020(95)00592-V

Synthesis of redox derivatives of lysine and their use in solid-phase synthesis of a light-harvesting peptide

Redox-active amino acids were synthesized for incorporation into peptide assemblies to study photoinitiated electron or energy transfer. 4′-Methyl-2,2′-bipyridine-4-carboxylic acid was obtained in 72% yield by consecutive SeO2 and Ag2O oxidation without isolation of intermediates. The side chain ε-amino group of Boc-l-lysine methyl ester or γ-carboxyl group of Boc-l-glutamic acid α-methyl ester was coupled to a redox moiety (transition-metal chromophore, electron donor, electron acceptor, metal ligand, or triplet-energy transmitter) using 4-(dimethylamino)pyridine, (1-benzotriazoleoxy)tris(dimethylamino)phosphonium hexafluorophosphate, N-methylmorpholine, and 1-hydroxybenzotriazole. Use of one equivalent of 4-(dimethylamino)pyridine provided the amide coupling product in 80-97% isolated yield. Selective hydrolysis of the methyl esters with lithium hydroxide provided the redox Boc-amino acids in 70-98% yield. These redox modules are suitable for solid-phase assembly of light-harvesting peptides, as illustrated by the synthesis of the partially α-helical 11-residue redox triad that contains a phenothiazine electron donor, a ruthenium(II)tris(bipyridine) chromophore, and an anthraquinone electron acceptor. Upon laser excitation at 420 nm, the peptide triad underwent photoinduced electron transfer to create a charge-separated state with a lifetime of 53 ns and decayed with a first-order rate constant of 1.9 × 108 s-1. © 1995.

Authors
McCafferty, DG; Bishop, BM; Wall, CG; Hughes, SG; Mecklenberg, SL; Meyer, TJ; Erickson, BW
MLA Citation
McCafferty, DG, Bishop, BM, Wall, CG, Hughes, SG, Mecklenberg, SL, Meyer, TJ, and Erickson, BW. "Synthesis of redox derivatives of lysine and their use in solid-phase synthesis of a light-harvesting peptide." Tetrahedron 51.4 (1995): 1093-1106.
Source
scival
Published In
Tetrahedron
Volume
51
Issue
4
Publish Date
1995
Start Page
1093
End Page
1106

Spectroscopic study of electron transfer in a trifunctional lysine with anthraquinone as the electron acceptor

The transient properties of the redox-active amino acid dyads [Anq-Lys(RuIIb2m)2+-OCH3] (PF6)2 and [Boc-Lys-(RuIIb2m)2+-NH-prPTZ] (PF6)2 and the trifunctionalized amino acid [Anq-Lys(RuIIb2m)2+-NH-prPTZ] (PF6)2, where Anq is 9,10-anthraquinone-2-carbonyl, Lys is L-lysine, b is 2,2′-bipyridine, m is 4′-methyl-2,2′-bipyridine-4-carbonyl, and prPTZ is 3-(10H-phenothiazine-10)propyl, were examined in CH3CN after nanosecond laser excitation. This series of redox-active assemblies was prepared by attaching derivatives of the ruthenium tris(bipyridyl) chromophore, the electron donor phenothiazine, and/or the electron acceptor anthraquinone to L-lysine with amide bonds. Emission from the chromophore was efficiently quenched (>95%) by the attached donors or acceptors in all three cases. Irradiation of [Anq-Lys(RuIIb2m)2+-NH-PrPTZ] with 420-nm, 4-ns laser pulses resulted in net electron transfer from prPTZ to Anq, mediated by the metal-to-ligand charge-transfer (MLCT) excited state of the ruthenium chromophore, as observed by nanosecond transient absorption and time-resolved resonance Raman spectroscopies. The resulting redox-separated state, [(Anq•-)-Lys(RuIIb2m) 2+-NH-(prPTZ•+)], at 1.54 eV, was formed with a quantum efficiency of 26% at its maximum appearance and persisted for 174 ns in CH3CN at 25 °C. © 1994 American Chemical Society.

Authors
Mecklenburg, SL; McCafferty, DG; Schoonover, JR; Peek, BM; Erickson, BW; Meyer, TJ
MLA Citation
Mecklenburg, SL, McCafferty, DG, Schoonover, JR, Peek, BM, Erickson, BW, and Meyer, TJ. "Spectroscopic study of electron transfer in a trifunctional lysine with anthraquinone as the electron acceptor." Inorganic Chemistry 33.13 (1994): 2974-2983.
Source
scival
Published In
Inorganic Chemistry
Volume
33
Issue
13
Publish Date
1994
Start Page
2974
End Page
2983

Photoinduced electron transfer in amino acid assemblies

The preparation and photophysical characterization of a of redox-active lysines and related model compounds based on polypyridyl ruthenium complexes are described. Donor-chromophore-acceptor triad 1, [PTZpn-Lys(RuIIb2m)2+-NH-prPQ2+] (PF6-)4 (see below), by of a bipyridyl caromophore (RuIIb2m, where b = 2,2′-bipyridine, m = 4′-methyl-2,2′-bipyridyl-4′-carbonyl), an electron donor (phenothiazine, PTZ), and an (paraquat, PQ2+) on a (Lys) scaffold utilizing bonds. This derivatized amiono acid exihibited efficient (>95%) quenching of the ruthenium metal-to-ligand charge-transfer (MLCT) excited state upon irradiation with a 420-nm laser pulse in CH3CN. The resulting state, [(PTZpn+)-Lys(RuIIb2m) 2+-NH-(prPQ+)]) 1.17 eV and lived for 108 ns (k = 9.26 × 106 s-1) as observed by transient absorption spectrosoopy. Also studied was a of related model systems that included model chroaophores, simple chromophore-quencher dyads linked by amide bonds, and chromophore-queneher dyads on lysine. An account of the of kinetic behavior of these system including triad 1 and a discussion of factors that influence the lifetime of the redox-separated states, their efficiency of formation, their energy storage ability are presented.

Authors
Mecklenburg, SL; Peek, BM; Schoonover, JR; McCafferty, DG; Wall, CG; Erickson, BW; Meyer, TJ
MLA Citation
Mecklenburg, SL, Peek, BM, Schoonover, JR, McCafferty, DG, Wall, CG, Erickson, BW, and Meyer, TJ. "Photoinduced electron transfer in amino acid assemblies." Journal of the American Chemical Society 115.13 (1993): 5479-5495.
Source
scival
Published In
Journal of the American Chemical Society
Volume
115
Issue
13
Publish Date
1993
Start Page
5479
End Page
5495
Show More