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Oas, Terrence Gilbert

Overview:

Our laboratory is primarily interested in the mechanisms of protein folding. We use nuclear magnetic resonance (NMR) and other types of spectroscopy to study the solution structure, stability and folding reactions of small protein models. These include monomeric λ repressor, the B domain of protein A (BdpA) and various regulator of G-protein signalling (RGS) domains. Our biophysical studies are used to inform our investigations of the role of folding mechanism in the function of proteins in the cell. For example, a naturally occuring cancer-causing mutation in the RGS domain of axin appears to lower the thermodynamic stability of the domain. We are developing methods to compensate for such destabilizing mutations, thereby restoring normal function to the protein.We are also developing computational models of protein folding as a way to better understand the mechanisms and as a tool in the design of new experiments.

Positions:

Professor of Biochemistry

Biochemistry
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1986

Ph.D. — University of Oregon

Grants:

Copper Homeostasis in Mammals

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
February 01, 2004
End Date
February 28, 2022

Role of protein A structure, folding kinetics and dynamics in S. aureus virulence

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2016
End Date
February 26, 2020

Structural Biology and Biophysics Training Program

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1994
End Date
September 30, 2015

New Console and Cold Probe for the Duke 600 MHz NMR Spectrometer System

Administered By
Radiology
AwardedBy
National Institutes of Health
Role
Major User
Start Date
June 15, 2013
End Date
June 14, 2014

Mechanistic Studies of Complex Protein Folding Reactions

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
December 01, 2008
End Date
November 30, 2013

SAXS System

Administered By
Pratt School of Engineering
AwardedBy
National Science Foundation
Role
Co Investigator
Start Date
August 01, 2012
End Date
July 31, 2013

Replacement Equipment Components for an 800 MHz NMR Spectrometer

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

Purchase of a Q-TOF LC/MS System

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

Duke PREP: Minority Recruitment into Biomedical Sciences

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Advisor
Start Date
August 01, 2003
End Date
July 31, 2008

Biophysical Studies of RNase P Protein Folding & Assembly

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2001
End Date
March 31, 2007

RNase P as a Model for Ribonucleoprotein Assembly

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2002
End Date
November 15, 2005

Mechanistic Studies of Protein Folding

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 1991
End Date
June 30, 2005

Folding Studies of Lambda Repressor Using Mutagenesis

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 1997
End Date
March 31, 2000

Nuclear Magnetic Resonance Studies Of Protein Folding

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
December 01, 1996
End Date
November 30, 1999

Molecular Biophysics Training Program

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1994
End Date
June 30, 1999

Structural Studies Of An Hiv-1 Inhibitory Peptide

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1993
End Date
March 31, 1997

Structural Studies Of Bpti Folding Mutants

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 1991
End Date
November 30, 1996

Structural Stuides Of An Hiv-1 Inhibitory Peptide

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1993
End Date
March 31, 1996

Structural Studies Of Bpti Folding Mutants

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 1994
End Date
December 31, 1995

Structural Studies Of Bpri Folding Mutants

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 1993
End Date
December 31, 1995

Circular Dichroism Spectrometer

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 1993
End Date
September 29, 1995
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Publications:

Spontaneous Unfolding-Refolding of Fibronectin Type III Domains Assayed by Thiol Exchange: THERMODYNAMIC STABILITY CORRELATES WITH RATES OF UNFOLDING RATHER THAN FOLDING.

Globular proteins are not permanently folded but spontaneously unfold and refold on time scales that can span orders of magnitude for different proteins. A longstanding debate in the protein-folding field is whether unfolding rates or folding rates correlate to the stability of a protein. In the present study, we have determined the unfolding and folding kinetics of 10 FNIII domains. FNIII domains are one of the most common protein folds and are present in 2% of animal proteins. FNIII domains are ideal for this study because they have an identical seven-strand β-sandwich structure, but they vary widely in sequence and thermodynamic stability. We assayed thermodynamic stability of each domain by equilibrium denaturation in urea. We then assayed the kinetics of domain opening and closing by a technique known as thiol exchange. For this we introduced a buried Cys at the identical location in each FNIII domain and measured the kinetics of labeling with DTNB over a range of urea concentrations. A global fit of the kinetics data gave the kinetics of spontaneous unfolding and refolding in zero urea. We found that the folding rates were relatively similar, ∼0.1-1 s-1, for the different domains. The unfolding rates varied widely and correlated with thermodynamic stability. Our study is the first to address this question using a set of domains that are structurally homologous but evolved with widely varying sequence identity and thermodynamic stability. These data add new evidence that thermodynamic stability correlates primarily with unfolding rate rather than folding rate. The study also has implications for the question of whether opening of FNIII domains contributes to the stretching of fibronectin matrix fibrils.

Authors
Shah, R; Ohashi, T; Erickson, HP; Oas, TG
MLA Citation
Shah, R, Ohashi, T, Erickson, HP, and Oas, TG. "Spontaneous Unfolding-Refolding of Fibronectin Type III Domains Assayed by Thiol Exchange: THERMODYNAMIC STABILITY CORRELATES WITH RATES OF UNFOLDING RATHER THAN FOLDING." The Journal of biological chemistry 292.3 (January 2017): 955-966.
Website
http://hdl.handle.net/10161/13915
PMID
27909052
Source
epmc
Published In
The Journal of biological chemistry
Volume
292
Issue
3
Publish Date
2017
Start Page
955
End Page
966
DOI
10.1074/jbc.m116.760371

Electrostatic Energetics of Bacillus subtilis Ribonuclease P Protein Determined by Nuclear Magnetic Resonance-Based Histidine pKa Measurements.

The pKa values of ionizable groups in proteins report the free energy of site-specific proton binding and provide a direct means of studying pH-dependent stability. We measured histidine pKa values (H3, H22, and H105) in the unfolded (U), intermediate (I), and sulfate-bound folded (F) states of RNase P protein, using an efficient and accurate nuclear magnetic resonance-monitored titration approach that utilizes internal reference compounds and a parametric fitting method. The three histidines in the sulfate-bound folded protein have pKa values depressed by 0.21 ± 0.01, 0.49 ± 0.01, and 1.00 ± 0.01 units, respectively, relative to that of the model compound N-acetyl-l-histidine methylamide. In the unliganded and unfolded protein, the pKa values are depressed relative to that of the model compound by 0.73 ± 0.02, 0.45 ± 0.02, and 0.68 ± 0.02 units, respectively. Above pH 5.5, H22 displays a separate resonance, which we have assigned to I, whose apparent pKa value is depressed by 1.03 ± 0.25 units, which is ∼0.5 units more than in either U or F. The depressed pKa values we observe are consistent with repulsive interactions between protonated histidine side chains and the net positive charge of the protein. However, the pKa differences between F and U are small for all three histidines, and they have little ionic strength dependence in F. Taken together, these observations suggest that unfavorable electrostatics alone do not account for the fact that RNase P protein is intrinsically unfolded in the absence of ligand. Multiple factors encoded in the P protein sequence account for its IUP property, which may play an important role in its function.

Authors
Mosley, PL; Daniels, KG; Oas, TG
MLA Citation
Mosley, PL, Daniels, KG, and Oas, TG. "Electrostatic Energetics of Bacillus subtilis Ribonuclease P Protein Determined by Nuclear Magnetic Resonance-Based Histidine pKa Measurements." Biochemistry 54.35 (September 2015): 5379-5388.
Website
http://hdl.handle.net/10161/10596
PMID
26267651
Source
epmc
Published In
Biochemistry
Volume
54
Issue
35
Publish Date
2015
Start Page
5379
End Page
5388
DOI
10.1021/acs.biochem.5b00138

Conformational kinetics reveals affinities of protein conformational states.

Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein's affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states.

Authors
Daniels, KG; Suo, Y; Oas, TG
MLA Citation
Daniels, KG, Suo, Y, and Oas, TG. "Conformational kinetics reveals affinities of protein conformational states." Proceedings of the National Academy of Sciences of the United States of America 112.30 (July 10, 2015): 9352-9357.
Website
http://hdl.handle.net/10161/10594
PMID
26162682
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
30
Publish Date
2015
Start Page
9352
End Page
9357
DOI
10.1073/pnas.1502084112

Suppression of conformational heterogeneity at a protein-protein interface.

Staphylococcal protein A (SpA) is an important virulence factor from Staphylococcus aureus responsible for the bacterium's evasion of the host immune system. SpA includes five small three-helix-bundle domains that can each bind with high affinity to many host proteins such as antibodies. The interaction between a SpA domain and the Fc fragment of IgG was partially elucidated previously in the crystal structure 1FC2. Although informative, the previous structure was not properly folded and left many substantial questions unanswered, such as a detailed description of the tertiary structure of SpA domains in complex with Fc and the structural changes that take place upon binding. Here we report the 2.3-Å structure of a fully folded SpA domain in complex with Fc. Our structure indicates that there are extensive structural rearrangements necessary for binding Fc, including a general reduction in SpA conformational heterogeneity, freezing out of polyrotameric interfacial residues, and displacement of a SpA side chain by an Fc side chain in a molecular-recognition pocket. Such a loss of conformational heterogeneity upon formation of the protein-protein interface may occur when SpA binds its multiple binding partners. Suppression of conformational heterogeneity may be an important structural paradigm in functionally plastic proteins.

Authors
Deis, LN; Wu, Q; Wang, Y; Qi, Y; Daniels, KG; Zhou, P; Oas, TG
MLA Citation
Deis, LN, Wu, Q, Wang, Y, Qi, Y, Daniels, KG, Zhou, P, and Oas, TG. "Suppression of conformational heterogeneity at a protein-protein interface." Proceedings of the National Academy of Sciences of the United States of America 112.29 (July 8, 2015): 9028-9033.
Website
http://hdl.handle.net/10161/10595
PMID
26157136
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
29
Publish Date
2015
Start Page
9028
End Page
9033
DOI
10.1073/pnas.1424724112

Visualizing the Inter-Domain Motions of a Pathogenic Protein using Sparse RDC Data

Authors
Qi, Y; Martin, JW; Yan, A; Thelot, F; Donald, BR; Oas, TG
MLA Citation
Qi, Y, Martin, JW, Yan, A, Thelot, F, Donald, BR, and Oas, TG. "Visualizing the Inter-Domain Motions of a Pathogenic Protein using Sparse RDC Data." BIOPHYSICAL JOURNAL 108.2 (January 27, 2015): 58A-58A.
Source
wos-lite
Published In
Biophysical Journal
Volume
108
Issue
2
Publish Date
2015
Start Page
58A
End Page
58A

Multiscale conformational heterogeneity in staphylococcal protein a: possible determinant of functional plasticity.

The Staphylococcus aureus virulence factor staphylococcal protein A (SpA) is a major contributor to bacterial evasion of the host immune system, through high-affinity binding to host proteins such as antibodies. SpA includes five small three-helix-bundle domains (E-D-A-B-C) separated by conserved flexible linkers. Prior attempts to crystallize individual domains in the absence of a binding partner have apparently been unsuccessful. There have also been no previous structures of tandem domains. Here we report the high-resolution crystal structures of a single C domain, and of two B domains connected by the conserved linker. Both structures exhibit extensive multiscale conformational heterogeneity, which required novel modeling protocols. Comparison of domain structures shows that helix1 orientation is especially heterogeneous, coordinated with changes in side chain conformational networks and contacting protein interfaces. This represents the kind of structural plasticity that could enable SpA to bind multiple partners.

Authors
Deis, LN; Pemble, CW; Qi, Y; Hagarman, A; Richardson, DC; Richardson, JS; Oas, TG
MLA Citation
Deis, LN, Pemble, CW, Qi, Y, Hagarman, A, Richardson, DC, Richardson, JS, and Oas, TG. "Multiscale conformational heterogeneity in staphylococcal protein a: possible determinant of functional plasticity." Structure (London, England : 1993) 22.10 (October 2014): 1467-1477.
Website
http://hdl.handle.net/10161/11167
PMID
25295398
Source
epmc
Published In
Structure
Volume
22
Issue
10
Publish Date
2014
Start Page
1467
End Page
1477
DOI
10.1016/j.str.2014.08.014

Visualizing the Inter-domain Motions of a Flexible Protein Using Continuous Models

Authors
Qi, Y; Martin, JW; Donald, BR; Oas, TG
MLA Citation
Qi, Y, Martin, JW, Donald, BR, and Oas, TG. "Visualizing the Inter-domain Motions of a Flexible Protein Using Continuous Models." July 2014.
Source
wos-lite
Published In
Protein Science
Volume
23
Publish Date
2014
Start Page
113
End Page
114

Developing Quantitative NMR Methods For Predicting Residue Specific Helicity Of MetO-lambda Unfolded State

Authors
Li, K; Hughes, R; Oas, TG
MLA Citation
Li, K, Hughes, R, and Oas, TG. "Developing Quantitative NMR Methods For Predicting Residue Specific Helicity Of MetO-lambda Unfolded State." July 2014.
Source
wos-lite
Published In
Protein Science
Volume
23
Publish Date
2014
Start Page
265
End Page
266

Determining the Rate of Unfolding and Refolding of FNIII Domains by Labeling Buried Cysteine

Authors
Shah, RS; Oas, TG; Erickson, HP
MLA Citation
Shah, RS, Oas, TG, and Erickson, HP. "Determining the Rate of Unfolding and Refolding of FNIII Domains by Labeling Buried Cysteine." January 28, 2014.
Source
wos-lite
Published In
Biophysical Journal
Volume
106
Issue
2
Publish Date
2014
Start Page
289A
End Page
289A

Ligand concentration regulates the pathways of coupled protein folding and binding.

Coupled ligand binding and conformational change plays a central role in biological regulation. Ligands often regulate protein function by modulating conformational dynamics, yet the order in which binding and conformational change occurs are often hotly debated. Here we show that the "conformational selection versus induced fit" distinction on which this debate is based is a false dichotomy because the mechanism depends on ligand concentration. Using the binding of pyrophosphate (PPi) to Bacillus subtilis RNase P protein as a model, we show that coupled reactions are best understood as a change in flux between competing pathways with distinct orders of binding and conformational change. The degree of partitioning through each pathway depends strongly on PPi concentration, with ligand binding redistributing the conformational ensemble toward the folded state by both increasing folding rates and decreasing unfolding rates. These results indicate that ligand binding induces marked and varied changes in protein conformational dynamics, and that the order of binding and conformational change is ligand concentration dependent.

Authors
Daniels, KG; Tonthat, NK; McClure, DR; Chang, Y-C; Liu, X; Schumacher, MA; Fierke, CA; Schmidler, SC; Oas, TG
MLA Citation
Daniels, KG, Tonthat, NK, McClure, DR, Chang, Y-C, Liu, X, Schumacher, MA, Fierke, CA, Schmidler, SC, and Oas, TG. "Ligand concentration regulates the pathways of coupled protein folding and binding." J Am Chem Soc 136.3 (January 22, 2014): 822-825.
PMID
24364358
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
136
Issue
3
Publish Date
2014
Start Page
822
End Page
825
DOI
10.1021/ja4086726

The statistical conformation of a highly flexible protein: Small-angle x-ray scattering of S. aureus protein A

Summary Staphylococcal protein A (SpA) is a multidomain protein consisting of five globular IgG binding domains separated by a conserved six- to nine-residue flexible linker. We collected SAXS data on the N-terminal protein-binding half of SpA (SpA-N) and constructs consisting of one to five domain modules in order to determine statistical conformation of this important S. aureus virulence factor. We fit the SAXS data to a scattering function based on a new polymer physics model, which provides an analytical description of the SpA-N statistical conformation. We describe a protocol for systematically determining the appropriate level of modeling to fit a SAXS data set based on goodness of fit and whether the addition of parameters improves it. In the case of SpA-N, the analytical polymer physics description provides a depiction of the statistical conformation of a flexible protein that, while lacking atomistic detail, properly reflects the information content of the data. © 2014 Elsevier Ltd.

Authors
Capp, JA; Hagarman, A; Richardson, DC; Oas, TG
MLA Citation
Capp, JA, Hagarman, A, Richardson, DC, and Oas, TG. "The statistical conformation of a highly flexible protein: Small-angle x-ray scattering of S. aureus protein A." Structure 22.8 (2014): 1184-1195.
Website
http://hdl.handle.net/10161/11168
Source
scival
Published In
Structure
Volume
22
Issue
8
Publish Date
2014
Start Page
1184
End Page
1195
DOI
10.1016/j.str.2014.06.011

Mapping mechanical properties of organic thin films by force-modulation microscopy in aqueous media.

The mechanical properties of organic and biomolecular thin films on surfaces play an important role in a broad range of applications. Although force-modulation microscopy (FMM) is used to map the apparent elastic properties of such films with high lateral resolution in air, it has rarely been applied in aqueous media. In this letter we describe the use of FMM to map the apparent elastic properties of self-assembled monolayers and end-tethered protein thin films in aqueous media. Furthermore, we describe a simple analysis of the contact mechanics that enables the selection of FMM imaging parameters and thus yields a reliable interpretation of the FMM image contrast.

Authors
Zhang, J; Parlak, Z; Bowers, CM; Oas, T; Zauscher, S
MLA Citation
Zhang, J, Parlak, Z, Bowers, CM, Oas, T, and Zauscher, S. "Mapping mechanical properties of organic thin films by force-modulation microscopy in aqueous media." Beilstein J Nanotechnol 3 (2012): 464-474.
PMID
23019540
Source
pubmed
Published In
Beilstein Journal of Nanotechnology
Volume
3
Publish Date
2012
Start Page
464
End Page
474
DOI
10.3762/bjnano.3.53

Multiple ligand-specific conformations of the β2-adrenergic receptor.

Seven-transmembrane receptors (7TMRs), also called G protein-coupled receptors (GPCRs), represent the largest class of drug targets, and they can signal through several distinct mechanisms including those mediated by G proteins and the multifunctional adaptor proteins β-arrestins. Moreover, several receptor ligands with differential efficacies toward these distinct signaling pathways have been identified. However, the structural basis and mechanism underlying this 'biased agonism' remains largely unknown. Here, we develop a quantitative mass spectrometry strategy that measures specific reactivities of individual side chains to investigate dynamic conformational changes in the β(2)-adrenergic receptor occupied by nine functionally distinct ligands. Unexpectedly, only a minority of residues showed reactivity patterns consistent with classical agonism, whereas the majority showed distinct patterns of reactivity even between functionally similar ligands. These findings demonstrate, contrary to two-state models for receptor activity, that there is significant variability in receptor conformations induced by different ligands, which has significant implications for the design of new therapeutic agents.

Authors
Kahsai, AW; Xiao, K; Rajagopal, S; Ahn, S; Shukla, AK; Sun, J; Oas, TG; Lefkowitz, RJ
MLA Citation
Kahsai, AW, Xiao, K, Rajagopal, S, Ahn, S, Shukla, AK, Sun, J, Oas, TG, and Lefkowitz, RJ. "Multiple ligand-specific conformations of the β2-adrenergic receptor. (Published online)" Nat Chem Biol 7.10 (August 21, 2011): 692-700.
PMID
21857662
Source
pubmed
Published In
Nature Chemical Biology
Volume
7
Issue
10
Publish Date
2011
Start Page
692
End Page
700
DOI
10.1038/nchembio.634

A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity.

Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High-throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity, which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol-reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand-binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose-binding protein engineered to contain single, protected cysteines. These straightforward, information-rich experiments are likely to find applications in protein engineering and functional genomics.

Authors
Isom, DG; Marguet, PR; Oas, TG; Hellinga, HW
MLA Citation
Isom, DG, Marguet, PR, Oas, TG, and Hellinga, HW. "A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity." Proteins 79.4 (April 2011): 1034-1047.
PMID
21387407
Source
pubmed
Published In
Proteins: Structure, Function and Bioinformatics
Volume
79
Issue
4
Publish Date
2011
Start Page
1034
End Page
1047
DOI
10.1002/prot.22932

Probing the folding intermediate of Bacillus subtilis RNase P protein by nuclear magnetic resonance.

Protein folding intermediates are often imperative for overall folding processes and consequent biological functions. However, the low population and transient nature of the intermediate states often hinder their biochemical and biophysical characterization. Previous studies have demonstrated that Bacillus subtilis ribonuclease P protein (P protein) is conformationally heterogeneous and folds with multiphasic kinetics, indicating the presence of an equilibrium and kinetic intermediate in its folding mechanism. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to study the ensemble corresponding to this intermediate (I). The results indicate that the N-terminal and C-terminal helical regions are mostly unfolded in I. 1H−15N heteronuclear single-quantum coherence NMR spectra collected as a function of pH suggest that the protonation of His 22 may play a major role in the energetics of the equilibria among the unfolded, intermediate, and folded state ensembles of P protein. NMR paramagnetic relaxation enhancement experiments were also used to locate the small anion binding sites in both the intermediate and folded ensembles. The results for the folded protein are consistent with the previously modeled binding regions. These structural insights suggest a possible role for I in the RNase P holoenzyme assembly process.

Authors
Chang, Y-C; Franch, WR; Oas, TG
MLA Citation
Chang, Y-C, Franch, WR, and Oas, TG. "Probing the folding intermediate of Bacillus subtilis RNase P protein by nuclear magnetic resonance." Biochemistry 49.44 (November 9, 2010): 9428-9437.
Website
http://hdl.handle.net/10161/4007
PMID
20843005
Source
pubmed
Published In
Biochemistry
Volume
49
Issue
44
Publish Date
2010
Start Page
9428
End Page
9437
DOI
10.1021/bi100287y

Osmolyte-induced folding of an intrinsically disordered protein: folding mechanism in the absence of ligand.

Understanding the interconversion between thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of the bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in this study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations exhibited multiphasic kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea during the titration to produce a urea-TMAO titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated, and the folding rate constants are relatively slow compared to those of intrinsically folded proteins similar in size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.

Authors
Chang, Y-C; Oas, TG
MLA Citation
Chang, Y-C, and Oas, TG. "Osmolyte-induced folding of an intrinsically disordered protein: folding mechanism in the absence of ligand." Biochemistry 49.25 (June 29, 2010): 5086-5096.
Website
http://hdl.handle.net/10161/4005
PMID
20476778
Source
pubmed
Published In
Biochemistry
Volume
49
Issue
25
Publish Date
2010
Start Page
5086
End Page
5096
DOI
10.1021/bi100222h

Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity.

The Gibbs free energy difference between native and unfolded states ("stability") is one of the fundamental characteristics of a protein. By exploiting the thermodynamic linkage between ligand binding and stability, interactions of a protein with small molecules, nucleic acids, or other proteins can be detected and quantified. Determination of protein stability can therefore provide a universal monitor of biochemical function. Yet, the use of stability measurements as a functional probe is underutilized, because such experiments traditionally require large amounts of protein and special instrumentation. Here we present the quantitative cysteine reactivity (QCR) technique to determine protein stabilities rapidly and accurately using only picomole quantities of material and readily accessible laboratory equipment. We demonstrate that QCR-derived stabilities can be used to measure ligand binding over a wide range of ligand concentrations and affinities. We anticipate that this technique will have broad applications in high-throughput protein engineering experiments and functional genomics.

Authors
Isom, DG; Vardy, E; Oas, TG; Hellinga, HW
MLA Citation
Isom, DG, Vardy, E, Oas, TG, and Hellinga, HW. "Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity." Proc Natl Acad Sci U S A 107.11 (March 16, 2010): 4908-4913.
PMID
20194783
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
107
Issue
11
Publish Date
2010
Start Page
4908
End Page
4913
DOI
10.1073/pnas.0910421107

Conformational selection or induced fit: a flux description of reaction mechanism.

The mechanism of ligand binding coupled to conformational changes in macromolecules has recently attracted considerable interest. The 2 limiting cases are the "induced fit" mechanism (binding first) or "conformational selection" (conformational change first). Described here are the criteria by which the sequence of events can be determined quantitatively. The relative importance of the 2 pathways is determined not by comparing rate constants (a common misconception) but instead by comparing the flux through each pathway. The simple rules for calculating flux in multistep mechanisms are described and then applied to 2 examples from the literature, neither of which has previously been analyzed using the concept of flux. The first example is the mechanism of conformational change in the binding of NADPH to dihydrofolate reductase. The second example is the mechanism of flavodoxin folding coupled to binding of its cofactor, flavin mononucleotide. In both cases, the mechanism switches from being dominated by the conformational selection pathway at low ligand concentration to induced fit at high ligand concentration. Over a wide range of conditions, a significant fraction of the flux occurs through both pathways. Such a mixed mechanism likely will be discovered for many cases of coupled conformational change and ligand binding when kinetic data are analyzed by using a flux-based approach.

Authors
Hammes, GG; Chang, Y-C; Oas, TG
MLA Citation
Hammes, GG, Chang, Y-C, and Oas, TG. "Conformational selection or induced fit: a flux description of reaction mechanism." Proc Natl Acad Sci U S A 106.33 (August 18, 2009): 13737-13741.
PMID
19666553
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
33
Publish Date
2009
Start Page
13737
End Page
13741
DOI
10.1073/pnas.0907195106

PHYS 26-When folding is coupled to binding: How do we tell which comes first?

Authors
Oas, TG
MLA Citation
Oas, TG. "PHYS 26-When folding is coupled to binding: How do we tell which comes first?." August 17, 2008.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
236
Publish Date
2008

Dynamics of backbone conformational heterogeneity in Bacillus subtilis ribonuclease P protein.

Interconversion of protein conformations is imperative to function, as evidenced by conformational changes associated with enzyme catalytic cycles, ligand binding and post-translational modifications. In this study, we used 15N NMR relaxation experiments to probe the fast (i.e., ps-ns) and slow (i.e., micros-ms) conformational dynamics of Bacillus subtilis ribonuclease P protein (P protein) in its folded state, bound to two sulfate anions. Using the Lipari-Szabo mapping method [Andrec, M., Montelione, G. T., and Levy, R. M. (2000) J. Biomol. NMR 18, 83-100] to interpret the data, we find evidence for P protein dynamics on the mus-ms time scale in the ensemble. The residues that exhibit these slow internal motions are found in regions that have been previously identified as part of the P protein-P RNA interface. These results suggest that structural flexibility within the P protein ensemble may be important for proper RNase P holoenzyme assembly and/or catalysis.

Authors
Henkels, CH; Chang, Y-C; Chamberlin, SI; Oas, TG
MLA Citation
Henkels, CH, Chang, Y-C, Chamberlin, SI, and Oas, TG. "Dynamics of backbone conformational heterogeneity in Bacillus subtilis ribonuclease P protein." Biochemistry 46.51 (December 25, 2007): 15062-15075.
PMID
18052200
Source
pubmed
Published In
Biochemistry
Volume
46
Issue
51
Publish Date
2007
Start Page
15062
End Page
15075
DOI
10.1021/bi701425n

Statistical estimation of statistical mechanical models: helix-coil theory and peptide helicity prediction.

Analysis of biopolymer sequences and structures generally adopts one of two approaches: use of detailed biophysical theoretical models of the system with experimentally-determined parameters, or largely empirical statistical models obtained by extracting parameters from large datasets. In this work, we demonstrate a merger of these two approaches using Bayesian statistics. We adopt a common biophysical model for local protein folding and peptide configuration, the helix-coil model. The parameters of this model are estimated by statistical fitting to a large dataset, using prior distributions based on experimental data. L(1)-norm shrinkage priors are applied to induce sparsity among the estimated parameters, resulting in a significantly simplified model. Formal statistical procedures for evaluating support in the data for previously proposed model extensions are presented. We demonstrate the advantages of this approach including improved prediction accuracy and quantification of prediction uncertainty, and discuss opportunities for statistical design of experiments. Our approach yields a 39% improvement in mean-squared predictive error over the current best algorithm for this problem. In the process we also provide an efficient recursive algorithm for exact calculation of ensemble helicity including sidechain interactions, and derive an explicit relation between homo- and heteropolymer helix-coil theories and Markov chains and (non-standard) hidden Markov models respectively, which has not appeared in the literature previously.

Authors
Schmidler, SC; Lucas, JE; Oas, TG
MLA Citation
Schmidler, SC, Lucas, JE, and Oas, TG. "Statistical estimation of statistical mechanical models: helix-coil theory and peptide helicity prediction." J Comput Biol 14.10 (December 2007): 1287-1310.
PMID
18047425
Source
pubmed
Published In
Journal of Computational Biology
Volume
14
Issue
10
Publish Date
2007
Start Page
1287
End Page
1310
DOI
10.1089/cmb.2007.0008

The active conformation of beta-arrestin1: direct evidence for the phosphate sensor in the N-domain and conformational differences in the active states of beta-arrestins1 and -2.

beta-Arrestins are multifunctional adaptor proteins that regulate seven transmembrane-spanning receptor (7TMR) desensitization and internalization and also initiate alternative signaling pathways. Studies have shown that beta-arrestins undergo a conformational change upon interaction with agonist-occupied, phosphorylated 7TMRs. Although conformational changes have been reported for visual arrestin and beta-arrestin2, these studies are not representative of conformational changes in beta-arrestin1. Accordingly, in this study, we determine conformational changes in beta-arrestin1 using limited tryptic proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis in the presence of a phosphopeptide derived from the C terminus of the V(2) vasopressin receptor (V(2)Rpp) or the corresponding unphosphorylated peptide (V(2)Rnp). V(2)Rpp binds specifically to beta-arrestin1 causing significant conformational changes, whereas V(2)Rnp does not alter the conformation of beta-arrestin1. Upon V(2)Rpp binding, we show that the previously shielded Arg(393) becomes accessible, which indicates release of the C terminus. Moreover, we show that Arg(285) becomes more accessible, and this residue is located in a region of beta-arrestin1 responsible for stabilization of its polar core. These two findings demonstrate "activation" of beta-arrestin1, and we also show a functional consequence of the release of the C terminus of beta-arrestin1 by enhanced clathrin binding. In addition, we show marked protection of the N-domain of beta-arrestin1 in the presence of V(2)Rpp, which is consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. A striking difference in conformational changes is observed in beta-arrestin1 when compared with beta-arrestin2, namely the flexibility of the interdomain hinge region. This study represents the first direct evidence that the "receptor-bound" conformations of beta-arrestins1 and 2 are different.

Authors
Nobles, KN; Guan, Z; Xiao, K; Oas, TG; Lefkowitz, RJ
MLA Citation
Nobles, KN, Guan, Z, Xiao, K, Oas, TG, and Lefkowitz, RJ. "The active conformation of beta-arrestin1: direct evidence for the phosphate sensor in the N-domain and conformational differences in the active states of beta-arrestins1 and -2." J Biol Chem 282.29 (July 20, 2007): 21370-21381.
PMID
17513300
Source
pubmed
Published In
The Journal of biological chemistry
Volume
282
Issue
29
Publish Date
2007
Start Page
21370
End Page
21381
DOI
10.1074/jbc.M611483200

Force-induced prolyl cis-trans isomerization in elastin-like polypeptides.

Elastin-like polypeptides (ELPs) are stimulus-responsive polymers that contain repeats of five amino acids, Val-Pro-Gly-Xaa-Gly (VPGXG), where Xaa is a guest residue that can be any amino acid with the exception of proline. While studying the conformational mechanics of ELPs over a range of solvent conditions by single-molecule force spectroscopy, we noticed that some force-extension curves showed temperature-independent, extensional transitions that could not be fitted with a freely jointed chain or worm-like chain model. Here we show that the observed molecular elongation results from the force-induced peptidyl-prolyl cis-trans isomerization in prolines, which are repeated every fifth residue in the main chain of ELPs. Control experiments with poly(L-proline) demonstrate the similarity of the conformational transition between poly(L-proline) and ELPs. In contrast, the force-extension behavior of poly(L-lysine) showed no deviation in the relevant force range. Force-extension curves in hysteresis experiments showed an elongational difference between extension and relaxation pathways that suggests that the cis conformational state of the prolines could be exhausted on the time scale of the experiment. We present further computational evidence for this mechanism by Monte Carlo simulation of the force-extension behavior using an elastically coupled, two-state model. We believe ours is the first demonstration of force-induced prolyl cis-trans isomerization in proline-containing polypeptides. Our results suggest that single-molecule force spectroscopy could provide an alternate means to assay this important conformational transition in polypeptides.

Authors
Valiaev, A; Lim, DW; Oas, TG; Chilkoti, A; Zauscher, S
MLA Citation
Valiaev, A, Lim, DW, Oas, TG, Chilkoti, A, and Zauscher, S. "Force-induced prolyl cis-trans isomerization in elastin-like polypeptides." J Am Chem Soc 129.20 (May 23, 2007): 6491-6497.
PMID
17469821
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
129
Issue
20
Publish Date
2007
Start Page
6491
End Page
6497
DOI
10.1021/ja070147r

Backbone dynamics of the monomeric lambda repressor denatured state ensemble under nondenaturing conditions.

Oxidizing two native methionine residues predominantly populates the denatured state of monomeric lambda repressor (MetO-lambdaLS) under nondenaturing conditions. NMR was used to characterize the secondary structure and dynamics of MetO-lambdaLS in standard phosphate buffer. 13Calpha and 1Halpha chemical shift indices reveal a region of significant helicity between residues 9 and 29. This helical content is further supported by the observation of medium-range amide NOEs. The remaining residues do not exhibit significant helicity as determined by NMR. We determined 15N relaxation parameters for 64 of 85 residues at 600 and 800 MHz. There are two distinct regions of reduced flexibility, residues 8-32 in the N-terminal third and residues 50-83 in the C-terminal third. The middle third, residues 33-50, has greater flexibility. We have analyzed the amplitude of the backbone motions in terms of the physical properties of the amino acids and conclude that conformational restriction of the backbone MetO-lambdaLS is due to nascent helix formation in the region corresponding to native helix 1. The bulkiness of amino acid residues in the C-terminal third leads to the potential for hydrophobic interactions, which is suggested by chemical exchange detected by the difference in spectral density J(0) at the two static magnetic fields. The more flexible middle region is the result of a predominance of small side chains in this region.

Authors
Chugha, P; Oas, TG
MLA Citation
Chugha, P, and Oas, TG. "Backbone dynamics of the monomeric lambda repressor denatured state ensemble under nondenaturing conditions." Biochemistry 46.5 (February 6, 2007): 1141-1151.
PMID
17260944
Source
pubmed
Published In
Biochemistry
Volume
46
Issue
5
Publish Date
2007
Start Page
1141
End Page
1151
DOI
10.1021/bi061371g

Folding mechanism of a multiple independently-folding domain protein: double B domain of protein A.

The antibody binding properties of staphylococcal protein A (SpA) can be attributed to the presence of five highly homologous domains (E, D, A, B, and C). Although the folding of the B domain of protein A (BdpA) is well-characterized, the folding behavior of this domain in the context of full-length SpA in the cell remains unexplored. The sequence of the B domain is 89 and 91% identical to those of domains A and C, respectively. We have fused B domain sequences (BBdpA) as a close approximation of the A-B or B-C portion of SpA. Circular dichroism and fluorescence-detected denaturation curves of BBdpA are experimentally indistinguishable from those of BdpA. The rate constants for folding and unfolding from NMR line shape analysis for the single- and double-domain proteins are the same within experimental uncertainties (+/-20%). These results support the designation of SpA as a multiple independently-folding domain (MIFD) protein. We develop a mathematical model that describes the folding thermodynamics and kinetics of MIFD proteins. The model depicts MIFD protein folding and unfolding as a parallel network and explicitly calculates the flux through all parallel pathways. These fluxes are combined to give a complete description of the global thermodynamics and kinetics of the folding and unfolding of MIFD proteins. The global rates for complete folding and unfolding of a MIFD protein and those of the individual domains depend on the stability of the protein. We show that the global unfolding rate of a MIFD protein may be many orders of magnitude slower than that of the constituent domains.

Authors
Arora, P; Hammes, GG; Oas, TG
MLA Citation
Arora, P, Hammes, GG, and Oas, TG. "Folding mechanism of a multiple independently-folding domain protein: double B domain of protein A." Biochemistry 45.40 (October 10, 2006): 12312-12324.
PMID
17014084
Source
pubmed
Published In
Biochemistry
Volume
45
Issue
40
Publish Date
2006
Start Page
12312
End Page
12324
DOI
10.1021/bi060923s

Ligation-state hydrogen exchange: coupled binding and folding equilibria in ribonuclease P protein.

Bacillus subtilis ribonuclease P protein (P protein) is predominantly unfolded (D) at physiological pH and low ionic strength; however, small molecule anionic ligands (e.g., sulfate) directly bind to and stabilize the folded state (NL2). Because the D + 2L <--> NL2 transition is experimentally two-state, high-energy states such as the singly bound, folded species (NL) and the unliganded folded species (N) are generally difficult to detect at equilibrium. To study the conformational properties of these ensembles, NMR-detected amide hydrogen exchange (HX) rates of P protein were measured at four sulfate (i.e., ligand) concentrations, a method we denote "ligation-state hydrogen exchange". The ligand concentration dependence of the HX rate of 47 residues was fit to a model with four possible HX pathways, corresponding to the local and/or global opening reactions from NL2 and NL, the local opening of N, and the global opening of N to D. Data analysis permits the calculation of the residue-specific free energy of opening from each ensemble as well as the fractional amide HX flux through each pathway. Results indicate that the predominant route of HX is through the NL and N states, which represent only 0.45% and 0.0005% of the total protein population in 20 mM sodium sulfate, respectively. Despite the low population of N, a region of protected amides was identified. Therefore, exchange through unliganded forms must be accounted for prior to the interpretation of HX-based protein-interaction studies. We offer a simple test to determine if HX occurs through the liganded or unliganded form.

Authors
Henkels, CH; Oas, TG
MLA Citation
Henkels, CH, and Oas, TG. "Ligation-state hydrogen exchange: coupled binding and folding equilibria in ribonuclease P protein." J Am Chem Soc 128.24 (June 21, 2006): 7772-7781.
PMID
16771491
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
128
Issue
24
Publish Date
2006
Start Page
7772
End Page
7781
DOI
10.1021/ja057279+

Ligation-state hydrogen exchange: coupled binding and folding equilibria in ribonuclease P protein.

Bacillus subtilis ribonuclease P protein (P protein) is predominantly unfolded (D) at physiological pH and low ionic strength; however, small molecule anionic ligands (e.g., sulfate) directly bind to and stabilize the folded state (NL2). Because the D + 2L NL2 transition is experimentally two-state, high-energy states such as the singly bound, folded species (NL) and the unliganded folded species (N) are generally difficult to detect at equilibrium. To study the conformational properties of these ensembles, NMR-detected amide hydrogen exchange (HX) rates of P protein were measured at four sulfate (i.e., ligand) concentrations, a method we denote "ligation-state hydrogen exchange". The ligand concentration dependence of the HX rate of 47 residues was fit to a model with four possible HX pathways, corresponding to the local and/or global opening reactions from NL2 and NL, the local opening of N, and the global opening of N to D. Data analysis permits the calculation of the residue-specific free energy of opening from each ensemble as well as the fractional amide HX flux through each pathway. Results indicate that the predominant route of HX is through the NL and N states, which represent only 0.45% and 0.0005% of the total protein population in 20 mM sodium sulfate, respectively. Despite the low population of N, a region of protected amides was identified. Therefore, exchange through unliganded forms must be accounted for prior to the interpretation of HX-based protein-interaction studies. We offer a simple test to determine if HX occurs through the liganded or unliganded form.

Authors
Henkels, CH; Oas, TG
MLA Citation
Henkels, CH, and Oas, TG. "Ligation-state hydrogen exchange: coupled binding and folding equilibria in ribonuclease P protein." Journal of the American Chemical Society 128.24 (June 2006): 7772-7781. (Academic Article)
Source
manual
Published In
Journal of the American Chemical Society
Volume
128
Issue
24
Publish Date
2006
Start Page
7772
End Page
7781
DOI
10.1021/ja057279

A statistical thermodynamic model of the protein ensemble.

Authors
Hilser, VJ; García Moreno, B; Oas, ETG; Kapp, G; Whitten, ST
MLA Citation
Hilser, VJ, García Moreno, B, Oas, ETG, Kapp, G, and Whitten, ST. "A statistical thermodynamic model of the protein ensemble." Chemical reviews 106.5 (May 2006): 1545-1558. (Academic Article)
Source
manual
Published In
Chemical Reviews
Volume
106
Issue
5
Publish Date
2006
Start Page
1545
End Page
1558
DOI
10.1021/cr040423

A statistical thermodynamic model of the protein ensemble.

Authors
Hilser, VJ; García-Moreno E, B; Oas, TG; Kapp, G; Whitten, ST
MLA Citation
Hilser, VJ, García-Moreno E, B, Oas, TG, Kapp, G, and Whitten, ST. "A statistical thermodynamic model of the protein ensemble." Chem Rev 106.5 (May 2006): 1545-1558. (Review)
PMID
16683744
Source
pubmed
Published In
Chemical Reviews
Volume
106
Issue
5
Publish Date
2006
Start Page
1545
End Page
1558
DOI
10.1021/cr040423+

Conformational changes in beta-arrestin1: The importance of beta-arrestin1's N-domain

Authors
Nobles, KN; Guan, ZQ; Xiao, KH; Oas, TG; Lefkowitz, RJ
MLA Citation
Nobles, KN, Guan, ZQ, Xiao, KH, Oas, TG, and Lefkowitz, RJ. "Conformational changes in beta-arrestin1: The importance of beta-arrestin1's N-domain." March 6, 2006.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
20
Issue
4
Publish Date
2006
Start Page
A114
End Page
A114

Methionine oxidation of monomeric lambda repressor: the denatured state ensemble under nondenaturing conditions.

Although poorly understood, the properties of the denatured state ensemble are critical to the thermodynamics and the kinetics of protein folding. The most relevant conformations to cellular protein folding are the ones populated under physiological conditions. To avoid the problem of low expression that is seen with unstable variants, we used methionine oxidation to destabilize monomeric lambda repressor and predominantly populate the denatured state under nondenaturing buffer conditions. The denatured ensemble populated under these conditions comprises conformations that are compact. Analytical ultracentrifugation sedimentation velocity experiments indicate a small increase in Stokes radius over that of the native state. A significant degree of alpha-helical structure in these conformations is detected by far-UV circular dichroism, and some tertiary interactions are suggested by near-UV circular dichroism. The characteristics of the denatured state populated by methionine oxidation in nondenaturing buffer are very different from those found in chemical denaturant.

Authors
Chugha, P; Sage, HJ; Oas, TG
MLA Citation
Chugha, P, Sage, HJ, and Oas, TG. "Methionine oxidation of monomeric lambda repressor: the denatured state ensemble under nondenaturing conditions." Protein Sci 15.3 (March 2006): 533-542.
PMID
16452618
Source
pubmed
Published In
Protein Science
Volume
15
Issue
3
Publish Date
2006
Start Page
533
End Page
542
DOI
10.1110/ps.051856406

Thermodynamic characterization of the osmolyte- and ligand-folded states of Bacillus subtilis ribonuclease P protein.

In Bacillus subtilis, P protein is the noncatalytic component of ribonuclease P (RNase P) that is critical for achieving maximal nuclease activity under physiological conditions. P protein is predominantly unfolded (D) at neutral pH and low ionic strength; however, it folds upon the addition of sulfate anions (ligands) as well as the osmolyte trimethylamine N-oxide (TMAO) [Henkels, C. H., Kurz, J. C., Fierke, C. A., and Oas, T. G. (2001) Biochemistry 40, 2777-2789]. Since the molecular mechanisms that drive protein folding for these two solutes are different, CD thermal denaturation studies were employed to dissect the thermodynamics of protein unfolding from the two folded states. A global fit of the free-energy of TMAO-folded P protein versus [TMAO] and temperature yields T(S), DeltaH(S), and DeltaC(p) of unfolding for the poorly populated, unliganded, folded state (N) in the absence of TMAO. These thermodynamic parameters were used in the fit of the data from the coupled unfolding/ligand dissociation reaction to obtain the sulfate dissociation constant (K(d)) and the DeltaH and DeltaC(p) of dissociation. These fits yielded a DeltaC(p) of protein unfolding of 826 +/- 23 cal mol(-)(1) K(-)(1) and a DeltaC(p) of 1554 +/- 29 cal mol(-)(1) K(-)(1) for the coupled unfolding and dissociation reaction (NL(2) --> D + 2L). The apparent stoichiometry of sulfate binding is two, so the DeltaC(p) increment of ligand dissociation is 363 +/- 9 cal mol(-)(1) K(-)(1) per site. Because N and NL(2) appear to be structurally similar and therefore similarly solvated using standard biophysical analyses, we attribute a substantial portion of this DeltaC(p) increment to an increase in conformational heterogeneity coincident with the NL(2) --> N + 2L transition.

Authors
Henkels, CH; Oas, TG
MLA Citation
Henkels, CH, and Oas, TG. "Thermodynamic characterization of the osmolyte- and ligand-folded states of Bacillus subtilis ribonuclease P protein." Biochemistry 44.39 (October 4, 2005): 13014-13026.
PMID
16185070
Source
pubmed
Published In
Biochemistry
Volume
44
Issue
39
Publish Date
2005
Start Page
13014
End Page
13026
DOI
10.1021/bi0504613

Activation dependent conformational changes in beta-arrestin 2

Authors
Xiao, KH; Shenoy, SK; Nobles, K; Oas, TG; Lefkowitz, RJ
MLA Citation
Xiao, KH, Shenoy, SK, Nobles, K, Oas, TG, and Lefkowitz, RJ. "Activation dependent conformational changes in beta-arrestin 2." January 2005.
Source
wos-lite
Published In
Biophysical Journal
Volume
88
Issue
1
Publish Date
2005
Start Page
554A
End Page
554A

Kinetic role of helix caps in protein folding is context-dependent.

Secondary structure punctuation through specific backbone and side chain interactions at the beginning and end of alpha-helices has been proposed to play a key role in hierarchical protein folding mechanisms [Baldwin, R. L., and Rose, G. D. (1999) Trends Biochem. Sci. 24, 26-33; Presta, L. G., and Rose, G. D. (1988) Science 240, 1632-1641]. We have made site-specific substitutions in the N- and C-cap motifs of the 5-helix protein monomeric lambda repressor (lambda(6-85)) and have measured the rate constants for folding and unfolding of each variant. The consequences of C-cap changes are strongly context-dependent. When the C-cap was located at the chain terminus, changes had little energetic and no kinetic effect. However, substitutions in a C-cap at the boundary between helix 4 and the subsequent interhelical loop resulted in large changes to the stability and rate constants of the variant, showing a substantial kinetic role for this interior C-cap and suggesting a general kinetic role for interior helix C-caps. Statistical preferences tabulated separately for internal and terminal C-caps also show only weak residue preferences in terminal C-caps. This kinetic distinction between interior and terminal C-caps can explain the discrepancy between the near-absence of stability and kinetic effects seen for C-caps of isolated peptides versus the very strong C-cap effects seen for proteins in statistical sequence preferences and mutational energetics. Introduction of consensus, in-register N-capping motifs resulted in increased stability, accelerated folding, and slower unfolding. The kinetic measurements indicate that some of the new native-state capping interactions remain unformed in the transition state. The accelerated folding rates could result from helix stabilization without invoking a specific role for N-caps in the folding reaction.

Authors
Kapp, GT; Richardson, JS; Oas, TG
MLA Citation
Kapp, GT, Richardson, JS, and Oas, TG. "Kinetic role of helix caps in protein folding is context-dependent." Biochemistry 43.13 (April 6, 2004): 3814-3823.
PMID
15049688
Source
pubmed
Published In
Biochemistry
Volume
43
Issue
13
Publish Date
2004
Start Page
3814
End Page
3823
DOI
10.1021/bi035683k

Fast and faster: a designed variant of the B-domain of protein A folds in 3 microsec.

We have introduced the mutation glycine 29 to alanine, designed to increase the rate of protein folding, into the B-domain of protein A (BdpA). From NMR lineshape analysis, we find the G29A mutation increases the folding rate constant by threefold; the folding time is 3 microsec. Although wild-type BdpA folds extremely fast, simple-point mutations can still speed up the folding; thus, the folding rate is not evolutionarily maximized. The short folding time of G29A BdpA (the shortest time yet reported) makes it an attractive candidate for an all-atom molecular dynamics simulation that could potentially show a complete folding reaction starting from an extended chain. We also constructed a fluorescent variant of BdpA by mutating phenylalanine 13 to tryptophan, allowing fluorescence-based time-resolved temperature-jump measurements. Temperature jumps and NMR complement each other, and give a very complete picture of the folding kinetics.

Authors
Arora, P; Oas, TG; Myers, JK
MLA Citation
Arora, P, Oas, TG, and Myers, JK. "Fast and faster: a designed variant of the B-domain of protein A folds in 3 microsec." Protein Sci 13.4 (April 2004): 847-853.
PMID
15044721
Source
pubmed
Published In
Protein Science
Volume
13
Issue
4
Publish Date
2004
Start Page
847
End Page
853
DOI
10.1110/ps.03541304

Probing the folding and unfolding dynamics of secondary and tertiary structures in a three-helix bundle protein.

Fast relaxation kinetics studies of the B-domain of staphylococcal protein A were performed to characterize the folding and unfolding of this small three-helix bundle protein. The relaxation kinetics were initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The kinetics monitored within the amide I' absorbance of the polypeptide backbone exhibit two distinct kinetics phases with nanosecond and microsecond relaxation times. The fast kinetics relaxation time is close to the diffusion limits placed on protein folding reactions. The fast kinetics phase is dominated by the relaxation of the solvated helix (nu = 1632 cm(-1)), which reports on the fast relaxation of the individual helices. The slow kinetics phase follows the cooperative relaxation of the native helical bundle core that is monitored by both solvated (nu = 1632 cm(-1)) and buried helical IR bands (nu = 1652 cm(-1)). The folding rates of the slow kinetics phase calculated over an extended temperature range indicate that the core formation of this protein follows a pathway that is energetically downhill. The unfolding rates are much more strongly temperature-dependent indicating an activated process with a large energy barrier. These results provide significant insight into the primary process of protein folding and suggest that fast formation of helices can drive the folding of helical proteins.

Authors
Vu, DM; Myers, JK; Oas, TG; Dyer, RB
MLA Citation
Vu, DM, Myers, JK, Oas, TG, and Dyer, RB. "Probing the folding and unfolding dynamics of secondary and tertiary structures in a three-helix bundle protein." Biochemistry 43.12 (March 30, 2004): 3582-3589.
PMID
15035628
Source
pubmed
Published In
Biochemistry
Volume
43
Issue
12
Publish Date
2004
Start Page
3582
End Page
3589
DOI
10.1021/bi036203s

Microsecond folding dynamics of the F13W G29A mutant of the B domain of staphylococcal protein A by laser-induced temperature jump.

The small size (58 residues) and simple structure of the B domain of staphylococcal protein A (BdpA) have led to this domain being a paradigm for theoretical studies of folding. Experimental studies of the folding of BdpA have been limited by the rapidity of its folding kinetics. We report the folding kinetics of a fluorescent mutant of BdpA (G29A F13W), named F13W*, using nanosecond laser-induced temperature jump experiments. Automation of the apparatus has permitted large data sets to be acquired that provide excellent signal-to-noise ratio over a wide range of experimental conditions. By measuring the temperature and denaturant dependence of equilibrium and kinetic data for F13W*, we show that thermodynamic modeling of multidimensional equilibrium and kinetic surfaces is a robust method that allows reliable extrapolation of rate constants to regions of the folding landscape not directly accessible experimentally. The results reveal that F13W* is the fastest-folding protein of its size studied to date, with a maximum folding rate constant at 0 M guanidinium chloride and 45 degrees C of 249,000 s(-1). Assuming the single-exponential kinetics represent barrier-limited folding, these data limit the value for the preexponential factor for folding of this protein to at least approximately 2 x 10(6) s(-1).

Authors
Dimitriadis, G; Drysdale, A; Myers, JK; Arora, P; Radford, SE; Oas, TG; Smith, DA
MLA Citation
Dimitriadis, G, Drysdale, A, Myers, JK, Arora, P, Radford, SE, Oas, TG, and Smith, DA. "Microsecond folding dynamics of the F13W G29A mutant of the B domain of staphylococcal protein A by laser-induced temperature jump." Proc Natl Acad Sci U S A 101.11 (March 16, 2004): 3809-3814.
PMID
15007169
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
101
Issue
11
Publish Date
2004
Start Page
3809
End Page
3814
DOI
10.1073/pnas.0306433101

Identification of a residue critical for maintaining the functional conformation of BPTI.

The effects of amino acid replacements on the backbone dynamics of bovine pancreatic trypsin inhibitor (BPTI) were examined using 15N NMR relaxation experiments. Previous studies have shown that backbone amide groups within the trypsin-binding region of the wild-type protein undergo conformational exchange processes on the micros time scale, and that replacement of Tyr35 with Gly greatly increases the number of backbone atoms involved in such motions. In order to determine whether these mutational effects are specific to the replacement of this residue with Gly, six additional replacements were examined in the present study. In two of these, Tyr35 was replaced with either Ala or Leu, and the other four were single replacements of Tyr23, Phe33, Asn43 or Asn44, all of which are highly buried in the native structure and conserved in homologous proteins. The Y35A and Y35L mutants displayed dynamic properties very similar to those of the Y35G mutant, with the backbone segments including residues 10-19 and 32-44 undergoing motions revealed by enhanced 15N transverse relaxation rates. On the other hand, the Y23L, N43G and N44A substitutions caused almost no detectable changes in backbone dynamics, on either the ns-ps or ms-micros time scales, even though each of these replacements significantly destabilizes the native conformation. Replacement of Phe33 with Leu caused intermediate effects, with several residues that have previously been implicated in motions in the wild-type protein displaying enhanced transverse relaxation rates. These results demonstrate that destabilizing amino acid replacements can be accommodated in a native protein with dramatically different effects on conformational dynamics and that Tyr35 plays a particularly important role in defining the conformation of the trypsin-binding site of BPTI.

Authors
Hanson, WM; Beeser, SA; Oas, TG; Goldenberg, DP
MLA Citation
Hanson, WM, Beeser, SA, Oas, TG, and Goldenberg, DP. "Identification of a residue critical for maintaining the functional conformation of BPTI." J Mol Biol 333.2 (October 17, 2003): 425-441.
PMID
14529627
Source
pubmed
Published In
Journal of Molecular Biology
Volume
333
Issue
2
Publish Date
2003
Start Page
425
End Page
441

A general mass spectrometry-based assay for the quantitation of protein-ligand binding interactions in solution (vol 124, pg 10256, 2002)

Authors
Powell, KD; Ghaemmaghami, S; Wang, MZ; Ma, LY; Oas, TG; Fitzgerald, MC
MLA Citation
Powell, KD, Ghaemmaghami, S, Wang, MZ, Ma, LY, Oas, TG, and Fitzgerald, MC. "A general mass spectrometry-based assay for the quantitation of protein-ligand binding interactions in solution (vol 124, pg 10256, 2002)." JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 125.14 (April 9, 2003): 4398-4398.
Source
wos-lite
Published In
Journal of the American Chemical Society
Volume
125
Issue
14
Publish Date
2003
Start Page
4398
End Page
4398
DOI
10.1021/ja0251266

Fast events in protein folding: Relaxation dynamics of a small three-helix bundle protein.

Authors
Vu, DM; Myers, JK; Oas, TG; Dyer, RB
MLA Citation
Vu, DM, Myers, JK, Oas, TG, and Dyer, RB. "Fast events in protein folding: Relaxation dynamics of a small three-helix bundle protein." February 2003.
Source
wos-lite
Published In
Biophysical Journal
Volume
84
Issue
2
Publish Date
2003
Start Page
166A
End Page
166A

A general mass spectrometry-based assay for the quantitation of protein-ligand binding interactions in solution.

A new method that utilizes matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and exploits the hydrogen/deuterium (H/D) exchange properties of proteins was developed for measuring the thermodynamic properties of protein-ligand complexes in solution. Dissociation constants (Kd values) determined by the method for five model protein-ligand complexes that included those with small molecules, nucleic acids, peptides, and other proteins were generally in good agreement with Kd values measured by conventional methods. Important experimental advantages of the described method over existing methods include: the ability to make measurements in a high-throughput and automated fashion, the ability to make measurements using only picomole quantitities of protein, and the ability to analyze either purified or unpurified protein-ligand complexes.

Authors
Powell, KD; Ghaemmaghami, S; Wang, MZ; Ma, L; Oas, TG; Fitzgerald, MC
MLA Citation
Powell, KD, Ghaemmaghami, S, Wang, MZ, Ma, L, Oas, TG, and Fitzgerald, MC. "A general mass spectrometry-based assay for the quantitation of protein-ligand binding interactions in solution." J Am Chem Soc 124.35 (September 4, 2002): 10256-10257.
PMID
12197709
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
124
Issue
35
Publish Date
2002
Start Page
10256
End Page
10257

Mechanism of fast protein folding.

An explosion of in vitro experimental data on the folding of proteins has revealed many examples of folding in the millisecond or faster timescale, often occurring in the absence of stable intermediate states. We review experimental methods for measuring fast protein folding kinetics, and then discuss various analytical models used to interpret these data. Finally, we classify general mechanisms that have been proposed to explain fast protein folding into two catagories, heterogeneous and homogeneous, reflecting the nature of the transition state. One heterogeneous mechanism, the diffusion-collision mechanism, can be used to interpret experimental data for a number of proteins.

Authors
Myers, JK; Oas, TG
MLA Citation
Myers, JK, and Oas, TG. "Mechanism of fast protein folding." Annu Rev Biochem 71 (2002): 783-815. (Review)
PMID
12045111
Source
pubmed
Published In
Annual Review of Biochemistry
Volume
71
Publish Date
2002
Start Page
783
End Page
815
DOI
10.1146/annurev.biochem.71.110601.135346

Quantitative protein stability measurement in living bacteria

Hydrogen exchange detected by MALDI mass spectrometry was used to measure the thermodynamic stability of proteins. The stability of the N-terminal domain measured in vitro, qualitatively correlated with its degradation because the folding kinetics of the protein was in rapid pre-equilibrium relative to its proteolysis. Some of the hydrogen atoms in peptides and proteins were labile to exchange with the surrounding solvent and a number of these exchangeable hydrogens were protected by the native conformation of the protein. The results show that the measured in vivo stability is 6.7 ± 0.13 kcal/mol and the in vitro stability of the protein obtained by performing H/D exchange on lysed bacteria at pH 8 is 6.7 ± 0.2 kcal/mol.

Authors
Ghaemmaghami, S; Fitzgerald, MC; Oas, TG
MLA Citation
Ghaemmaghami, S, Fitzgerald, MC, and Oas, TG. "Quantitative protein stability measurement in living bacteria." Proceedings 50th ASMS Conference on Mass Spectrometry and Allied Topics (2002): 109-110.
Source
scival
Published In
Proceedings 50th ASMS Conference on Mass Spectrometry and Allied Topics
Publish Date
2002
Start Page
109
End Page
110

A general mass spectrometry-based method for the quantitation of protein-ligand binding interactions in solution

The protein-ligand binding interactions in solution were investigated using mass spectrometry. The H/D exchange and mass spectrometry-based technique termed as stability of unpurified proteins by rates of H/D exchange (SUPREX) was developed to evaluate thermodynamic stability of proteins. The analysis was initiated by 10-fold dilution of stock solution of protein in series of deuterated exchange buffers which varied in concentration of deuterated guanidine. The results show that increased H/D time shifted the SUPREX curve transition midpoint to lower denaturant.

Authors
Fitzgerald, MC; Powell, KD; Wang, MZ; Ma, L; Ghaemmaghami, S; Oas, TG
MLA Citation
Fitzgerald, MC, Powell, KD, Wang, MZ, Ma, L, Ghaemmaghami, S, and Oas, TG. "A general mass spectrometry-based method for the quantitation of protein-ligand binding interactions in solution." Proceedings 50th ASMS Conference on Mass Spectrometry and Allied Topics (2002): 395-396.
Source
scival
Published In
Proceedings 50th ASMS Conference on Mass Spectrometry and Allied Topics
Publish Date
2002
Start Page
395
End Page
396

Quantitative protein stability measurement in vivo.

The equilibrium between the native and denatured states of a protein can be key to its function and regulation. Traditionally, the folding equilibrium constant has been measured in vitro using purified protein and simple buffers. However, the biological environment of proteins can differ from these in vitro conditions in ways that could significantly perturb stability. Here, we present the first quantitative comparison between the stability of a protein in vitro and in the cytoplasm of Escherichia coli using amide hydrogen exchange detected by MALDI mass spectrometry (SUPREX). The results indicate that the thermodynamic stability of monomeric lambda repressor within the cell is the same as its stability measured in a simple buffer in vitro. However, when the E. coli are placed in a hyperosmotic environment, the in vivo stability is greatly enhanced. The in vivo SUPREX method provides a general and quantitative way to measure protein stabilities in the cell and will be useful for applications where intracellular stability information provides important biological insights.

Authors
Ghaemmaghami, S; Oas, TG
MLA Citation
Ghaemmaghami, S, and Oas, TG. "Quantitative protein stability measurement in vivo." Nat Struct Biol 8.10 (October 2001): 879-882.
PMID
11573094
Source
pubmed
Published In
Nature Structural Biology
Volume
8
Issue
10
Publish Date
2001
Start Page
879
End Page
882
DOI
10.1038/nsb1001-879

Phosphorylation of RNA polymerase II CTD fragments results in tight binding to the WW domain from the yeast prolyl isomerase Ess1.

The yeast prolyl isomerase, Ess1, has recently been shown to interact via its WW domain with the hyperphosphorylated form of the RNA polymerase II C-terminal domain (CTD). We have investigated folding of the Ess1 WW domain and its binding to peptides representing the CTD by circular dichroism and fluorescence. Ess1 WW folds and unfolds reversibly, but in the absence of ligand is only marginally stable with a melting temperature of 19 degrees C. The WW domain is stabilized by the addition of anionic ligands, namely, chloride, inorganic phosphate, phosphoserine, and phosphorylated CTD peptides. Dissociation constants were measured to be 70--100 microM for CTD peptides phosphorylated at one serine, and 16--21 microM for peptides with two or more phosphorylated serines. Weaker or no affinity was observed for nonphosphorylated CTD peptides. There is surprisingly little difference in the affinity for peptides phosphorylated at Ser 2 or Ser 5 of the consensus repeat, or for peptides with different patterns of multiple phosphorylation. The binding of Ess1 to phosphorylated CTD peptides is consistent with a model wherein the WW domain positions Ess1 to catalyze isomerization of the many pSer--Pro peptide bonds in the phosphorylated CTD. We suggest that cis/trans isomerization of prolyl peptide bonds plays a crucial role in CTD function during eukaryotic transcription.

Authors
Myers, JK; Morris, DP; Greenleaf, AL; Oas, TG
MLA Citation
Myers, JK, Morris, DP, Greenleaf, AL, and Oas, TG. "Phosphorylation of RNA polymerase II CTD fragments results in tight binding to the WW domain from the yeast prolyl isomerase Ess1." Biochemistry 40.29 (July 24, 2001): 8479-8486.
PMID
11456485
Source
pubmed
Published In
Biochemistry
Volume
40
Issue
29
Publish Date
2001
Start Page
8479
End Page
8486

Preorganized secondary structure as an important determinant of fast protein folding.

The folding and unfolding kinetics of the B-domain of staphylococcal protein A, a small three-helix bundle protein, were probed by NMR. The lineshape of a single histidine resonance was fit as a function of denaturant to give folding and unfolding rate constants. The B-domain folds extremely rapidly in a two-state manner, with a folding rate constant of 120,000 s-1, making it one of the fastest-folding proteins known. Diffusion-collision theory predicts folding and unfolding rate constants that are in good agreement with the experimental values. The apparent rate constant as a function of denaturant ('chevron plot') is predicted within an order of magnitude. Our results are consistent with a model whereby fast-folding proteins utilize a diffusion-collision mechanism, with the preorganization of one or more elements of secondary structure in the unfolded protein.

Authors
Myers, JK; Oas, TG
MLA Citation
Myers, JK, and Oas, TG. "Preorganized secondary structure as an important determinant of fast protein folding." Nat Struct Biol 8.6 (June 2001): 552-558.
PMID
11373626
Source
pubmed
Published In
Nature Structural Biology
Volume
8
Issue
6
Publish Date
2001
Start Page
552
End Page
558
DOI
10.1038/88626

Linked folding and anion binding of the Bacillus subtilis ribonuclease P protein.

Ribonuclease P (RNase P) is the endoribonuclease responsible for the 5'-maturation of precursor tRNA transcripts. In bacteria, RNase P is composed of a catalytic RNA subunit and an associated protein subunit that enhances the substrate specificity of the holoenzyme. We have initiated a study of the biophysical properties of the protein subunit from Bacillus subtilis RNase P (P protein) toward the goal of understanding the thermodynamics of RNase P holoenzyme assembly. The P protein is predominantly unfolded in 10 mM sodium cacodylate at neutral pH based on circular dichroism and NMR studies and therefore has several characteristics typical of "intrinsically unstructured" proteins. Furthermore, the P protein folds to its native alpha/beta structure upon addition of various small molecule anions. Anion-induced folding is best attributed to the binding of these anions to the folded state of the protein, and a model is presented which describes the observed tightly coupled folding and binding phenomena. The P protein also undergoes a cooperative folding transition upon addition of the osmolyte trimethylamine N-oxide (TMAO). The equilibrium constant of folding (K(fold)) at 37 degrees C for the P protein was determined to be 0.0071 +/- 0.0005 using a two-state folding model to describe the TMAO titration data. Thus, the folding and binding equilibria observed in the anion-induced folding of the P protein can be uncoupled to determine the intrinsic binding affinities (K(a)'s) of the anionic ligands. Evidence that the osmolyte-induced and the ligand-induced folded conformations of the P protein are structurally similar is also presented.

Authors
Henkels, CH; Kurz, JC; Fierke, CA; Oas, TG
MLA Citation
Henkels, CH, Kurz, JC, Fierke, CA, and Oas, TG. "Linked folding and anion binding of the Bacillus subtilis ribonuclease P protein." Biochemistry 40.9 (March 6, 2001): 2777-2789.
PMID
11258888
Source
pubmed
Published In
Biochemistry
Volume
40
Issue
9
Publish Date
2001
Start Page
2777
End Page
2789

A quantitative, high-throughput screen for protein stability.

In proteomic research, it is often necessary to screen a large number of polypeptides for the presence of stable structure. Described here is a technique (referred to as SUPREX, stability of unpurified proteins from rates of H/D exchange) for measuring the stability of proteins in a rapid, high-throughput fashion. The method uses hydrogen exchange to estimate the stability of microgram quantities of unpurified protein extracts by using matrix-assisted laser desorption/ionization MS. The stabilities of maltose binding protein and monomeric lambda repressor variants determined by SUPREX agree well with stability data obtained from conventional CD denaturation of purified protein. The method also can detect the change in stability caused by the binding of maltose to maltose binding protein. The results demonstrate the precision of the method over a wide range of stabilities.

Authors
Ghaemmaghami, S; Fitzgerald, MC; Oas, TG
MLA Citation
Ghaemmaghami, S, Fitzgerald, MC, and Oas, TG. "A quantitative, high-throughput screen for protein stability." Proc Natl Acad Sci U S A 97.15 (July 18, 2000): 8296-8301.
PMID
10890887
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
97
Issue
15
Publish Date
2000
Start Page
8296
End Page
8301
DOI
10.1073/pnas.140111397

Novel disulfide engineering in human carbonic anhydrase II using the PAIRWISE side-chain geometry database.

An analysis of the pairwise side-chain packing geometries of cysteine residues observed in high-resolution protein crystal structures indicates that cysteine pairs have pronounced orientational preferences due to the geometric constraints of disulfide bond formation. A potential function was generated from these observations and used to evaluate models for novel disulfide bonds in human carbonic anhydrase II (HCAII). Three double-cysteine variants of HCAII were purified and the effective concentrations of their thiol groups were determined by titrations with glutathione and dithiothreitol. The effects of the cysteine mutations on the native state structure and stability were characterized by circular dichroism, enzymatic activity, sulfonamide binding, and guanidine hydrochloride titration. These analyses indicate that the PAIRWISE potential is a good predictor of the strength of the disulfide bond itself, but the overall structural and thermodynamic effects on the protein are complicated by additional factors. In particular, the effects of cysteine substitutions on the native state and the stabilization of compact nonnative states by the disulfide can override any stabilizing effect of the cross-link.

Authors
Burton, RE; Hunt, JA; Fierke, CA; Oas, TG
MLA Citation
Burton, RE, Hunt, JA, Fierke, CA, and Oas, TG. "Novel disulfide engineering in human carbonic anhydrase II using the PAIRWISE side-chain geometry database." Protein Sci 9.4 (April 2000): 776-785.
PMID
10794421
Source
pubmed
Published In
Protein Science
Volume
9
Issue
4
Publish Date
2000
Start Page
776
End Page
785
DOI
10.1110/ps.9.4.776

Mechanism of folding fast: Experimental and theoretical studies of monomeric lambda repressor.

Authors
Oas, TG
MLA Citation
Oas, TG. "Mechanism of folding fast: Experimental and theoretical studies of monomeric lambda repressor." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 219 (March 26, 2000): U283-U283.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
219
Publish Date
2000
Start Page
U283
End Page
U283

Reinterpretation of GCN4-p1 folding kinetics: partial helix formation precedes dimerization in coiled coil folding.

The folding of coiled coil peptides has traditionally been interpreted in terms of native dimer and unfolded monomers. Calculations using AGADIR and experimental studies of fragments suggest that the monomers of the coiled coil peptide, GCN4-p1, contain significant residual helical structure. A simple model based on diffusion-collision theory predicts not only the measured folding rate within an order of magnitude, but also predicts remarkably well the effect of alanine to glyXcine mutations. We suggest that intrinsic helix stability is a major determinant of the folding rate of the GCN4 coiled coil.

Authors
Myers, JK; Oas, TG
MLA Citation
Myers, JK, and Oas, TG. "Reinterpretation of GCN4-p1 folding kinetics: partial helix formation precedes dimerization in coiled coil folding." J Mol Biol 289.2 (June 4, 1999): 205-209.
PMID
10366499
Source
pubmed
Published In
Journal of Molecular Biology
Volume
289
Issue
2
Publish Date
1999
Start Page
205
End Page
209
DOI
10.1006/jmbi.1999.2747

Contribution of a buried hydrogen bond to lambda repressor folding kinetics.

A hydrogen bond between the buried residues Asp 14 and Ser 77 in monomeric lambda repressor has been removed by mutation of these residues to alanine. Double mutant cycles show that the interaction stabilizes the native state of the protein by 1.5 kcal/mol. Removal of the interaction affects mainly the unfolding rates and not the folding rates, suggesting that this hydrogen bond is not substantially formed in the rate-limiting steps in the folding pathways of the protein. Mutations in two versions of lambda6-85, wild type and the faster folding G46A/G48A (WT), show similar effects. Diffusion-collision correctly predicts the behavior of WT but not of wild type. Our analysis suggests that folding of helix 3 is a crucial slow step along the various folding pathways and generally occurs before the formation of the 14-77 hydrogen bond. Experiments removing tertiary interactions, combined with experiments altering helical stability and diffusion-collision calculations, provide a strategy to unravel the folding mechanisms of small helical proteins.

Authors
Myers, JK; Oas, TG
MLA Citation
Myers, JK, and Oas, TG. "Contribution of a buried hydrogen bond to lambda repressor folding kinetics." Biochemistry 38.21 (May 25, 1999): 6761-6768.
PMID
10346896
Source
pubmed
Published In
Biochemistry
Volume
38
Issue
21
Publish Date
1999
Start Page
6761
End Page
6768
DOI
10.1021/bi990088x

Determinants of backbone dynamics in native BPTI: cooperative influence of the 14-38 disulfide and the Tyr35 side-chain.

15Nitrogen relaxation experiments were used to characterize the backbone dynamics of two modified forms of bovine pancreatic trypsin inhibitor (BPTI). In one form, the disulfide between Cys14 and Cys38 in the wild-type protein was selectively reduced and methylated to generate an analog of the final intermediate in the disulfide-coupled folding pathway. The second form was generated by similarly modifying a mutant protein in which Tyr35 was replaced with Gly (Y35G). For both selectively reduced proteins, the overall conformation of native BPTI was retained, and the relaxation data for these proteins were compared with those obtained previously with the native wild-type and Y35G proteins. Removing the disulfide from either protein had only small effects on the observed longitudinal relaxation rates (R1) or heteronuclear cross relaxation rates (nuclear Overhauser effect), suggesting that the 14-38 disulfide has little influence on the fast (ps to ns) backbone dynamics of either protein. In the wild-type protein, the pattern of residues undergoing slower (micros to ms) internal motions, reflected in unusually large transverse relaxation rates (R2), was also largely unaffected by the removal of this disulfide. It thus appears that the large R2 rates previously observed in native wild-type protein are not a direct consequence of isomerization of the 14-38 disulfide. In contrast with the wild-type protein, reducing the disulfide in Y35G BPTI significantly decreased the number of backbone amides displaying large R2 rates. In addition, the frequencies of the backbone motions in the modified protein, estimated from R2 values measured at multiple refocusing delays, appear to span a wider range than those seen in native Y35G BPTI. Together, these observations suggest that the slow internal motions in Y35G BPTI are more independent in the absence of the 14-38 disulfide and that formation of this bond may lead to a substantial loss of conformational entropy. These effects may account for the previous observation that the Y35G substitution greatly destabilizes the disulfide. The results also demonstrate that the disulfide and the buried side-chain influence the dynamics of the folded protein in a highly cooperative fashion, with the effects of removing either being much greater in the absence of the other.

Authors
Beeser, SA; Oas, TG; Goldenberg, DP
MLA Citation
Beeser, SA, Oas, TG, and Goldenberg, DP. "Determinants of backbone dynamics in native BPTI: cooperative influence of the 14-38 disulfide and the Tyr35 side-chain." J Mol Biol 284.5 (December 18, 1998): 1581-1596.
PMID
9878372
Source
pubmed
Published In
Journal of Molecular Biology
Volume
284
Issue
5
Publish Date
1998
Start Page
1581
End Page
1596
DOI
10.1006/jmbi.1998.2240

The structural distribution of cooperative interactions in proteins: analysis of the native state ensemble.

Cooperative interactions link the behavior of different amino acid residues within a protein molecule. As a result, the effects of chemical or physical perturbations to any given residue are propagated to other residues by an intricate network of interactions. Very often, amino acids "sense" the effects of perturbations occurring at very distant locations in the protein molecule. In these studies, we have investigated by computer simulation the structural distribution of those interactions. We show here that cooperative interactions are not intrinsically bi-directional and that different residues play different roles within the intricate network of interactions existing in a protein. The effect of a perturbation to residue j on residue k is not necessarily equal to the effect of the same perturbation to residue k on residue j. In this paper, we introduce a computer algorithm aimed at mapping the network of cooperative interactions within a protein. This algorithm exhaustively performs single site thermodynamic mutations to each residue in the protein and examines the effects of those mutations on the distribution of conformational states. The algorithm has been applied to three different proteins (lambda repressor fragment 6-85, chymotrypsin inhibitor 2, and barnase). This algorithm accounts well for the observed behavior of these proteins.

Authors
Hilser, VJ; Dowdy, D; Oas, TG; Freire, E
MLA Citation
Hilser, VJ, Dowdy, D, Oas, TG, and Freire, E. "The structural distribution of cooperative interactions in proteins: analysis of the native state ensemble." Proc Natl Acad Sci U S A 95.17 (August 18, 1998): 9903-9908.
PMID
9707573
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
95
Issue
17
Publish Date
1998
Start Page
9903
End Page
9908

Folding kinetics of a fluorescent variant of monomeric lambda repressor.

A tryptophan-containing variant of monomeric lambda repressor has been made, and its folding kinetics were analyzed at 20 degreesC using fluorescence stopped-flow and dynamic NMR. Equilibrium denaturation curves obtained by circular dichroism, fluorescence, and NMR are superimposable. Stopped-flow analysis indicates that in the absence of denaturants the folding reaction is complete within the dead-time of the experiment. Within higher denaturant conditions, where the folding rate is slower, NMR and stopped-flow agree on the folding and unfolding rates of the protein. In 3.4 M urea and 1.8 M GdmCl, we show that the variant folds within 2 ms. Extrapolation indicates that the folding time is 20 micro(s) in the absence of denaturants. All folding and unfolding reactions displayed monoexponential kinetics, and no burst-phases were observed. In addition, the thermodynamic parameters Delta G and meq obtained from the kinetic analysis are consistent with the equilibrium experiments. The results support a two-state Dleft and right arrow N folding model.

Authors
Ghaemmaghami, S; Word, JM; Burton, RE; Richardson, JS; Oas, TG
MLA Citation
Ghaemmaghami, S, Word, JM, Burton, RE, Richardson, JS, and Oas, TG. "Folding kinetics of a fluorescent variant of monomeric lambda repressor." Biochemistry 37.25 (June 23, 1998): 9179-9185.
PMID
9636065
Source
pubmed
Published In
Biochemistry
Volume
37
Issue
25
Publish Date
1998
Start Page
9179
End Page
9185
DOI
10.1021/bi980356b

Protein folding dynamics: quantitative comparison between theory and experiment.

The development of a quantitative kinetic scheme is a central goal in mechanistic studies of biological phenomena. For fast-folding proteins, which lack experimentally observable kinetic intermediates, a quantitative kinetic scheme describing the order and rate of events during folding has yet to be developed. In the present study, the folding mechanism of monomeric lambda repressor is described using the diffusion-collision model and estimates of intrinsic alpha-helix propensities. The model accurately predicts the folding rates of the wild-type protein and five of eight previously studied Ala left and right arrow Gly variants and suggests that the folding mechanism is distributed among multiple pathways that are highly sensitive to the amino acid sequence. For example, the model predicts that the wild-type protein folds through a small number of pathways with a folding time of 260 micros. However, the folding of a variant (G46A/G48A) is predicted to fold through a large number of pathways with a folding time of 12 micros. Both folding times quantitatively agree with the experimental values at 37 degrees C extrapolated to 0 M denaturant. The quantitative nature of the diffusion-collision model allows for rigorous experimental tests of the theory.

Authors
Burton, RE; Myers, JK; Oas, TG
MLA Citation
Burton, RE, Myers, JK, and Oas, TG. "Protein folding dynamics: quantitative comparison between theory and experiment." Biochemistry 37.16 (April 21, 1998): 5337-5343.
PMID
9548914
Source
pubmed
Published In
Biochemistry
Volume
37
Issue
16
Publish Date
1998
Start Page
5337
End Page
5343
DOI
10.1021/bi980245c

Ultrafast folding of monomeric lambda repressor: Experimental characterization and accurate theoretical prediction of folding rates

Authors
Burton, RS; Myers, JK; Oas, TG
MLA Citation
Burton, RS, Myers, JK, and Oas, TG. "Ultrafast folding of monomeric lambda repressor: Experimental characterization and accurate theoretical prediction of folding rates." BIOPHYSICAL JOURNAL 74.2 (February 1998): A24-A24.
Source
wos-lite
Published In
Biophysical Journal
Volume
74
Issue
2
Publish Date
1998
Start Page
A24
End Page
A24

ALASKA: A Mathematica package for two-state kinetic analysis of protein folding reactions

A Mathematica package (ALASKA) has been developed to simplify the measurement of protein folding kinetics by analysis of 1H NMR lineshape analysis. This package reads NMR data in ASCII format and can simulate an aromatic 1 NMR spectrum with or without lineshape broadening from chemical exchange. We describe the analysis of a urea denaturation series of a fast-folding protein, the G46A/G48A variant of monomeric λ repressor.

Authors
Burton, RE; Busby, RS; Oas, TG
MLA Citation
Burton, RE, Busby, RS, and Oas, TG. "ALASKA: A Mathematica package for two-state kinetic analysis of protein folding reactions." Journal of Biomolecular NMR 11.3 (1998): 355-359.
Source
scival
Published In
Journal of Biomolecular NMR
Volume
11
Issue
3
Publish Date
1998
Start Page
355
End Page
359

Enhanced protein flexibility caused by a destabilizing amino acid replacement in BPTI.

A genetically engineered variant of bovine pancreatic trypsin inhibitor (Y35G BPTI) has been shown previously by X-ray crystallography to have a three-dimensional structure dramatically different from that of the wild-type protein, particularly in the protease-binding region of the molecule. Yet, the Y35G variant is a potent trypsin inhibitor. Described here are 15N NMR relaxation studies to compare the backbone dynamics of Y35G BPTI to those of the wild-type protein. The Tyr35 --> Gly substitution increased the transverse relaxation rates of more than one third of all backbone amide groups, but had little effect on the longitudinal relaxation rates, indicating that the substitution facilitates relatively slow backbone motions, estimated to be on the microsecond time-scale. The results indicate that the residues making up the trypsin-binding site undergo large and relatively slow conformational changes in solution, estimated to be on the 5 to 20 micros time-scale. It is thus likely that the crystal structure represents only one of multiple interconverting conformations in solution, only a fraction of which may be competent for binding trypsin. The large thermodynamic destabilization associated with this substitution may arise, in part, from a loss in cooperativity among the multiple stabilizing interactions that are normally favored by the highly ordered structure of the wild-type protein. These results suggest that fully understanding the effects of amino acid replacements on the functional and thermodynamic properties of proteins may often require analysis of the dynamic, as well as the structural, properties of altered proteins.

Authors
Beeser, SA; Goldenberg, DP; Oas, TG
MLA Citation
Beeser, SA, Goldenberg, DP, and Oas, TG. "Enhanced protein flexibility caused by a destabilizing amino acid replacement in BPTI." J Mol Biol 269.1 (May 30, 1997): 154-164.
PMID
9193007
Source
pubmed
Published In
Journal of Molecular Biology
Volume
269
Issue
1
Publish Date
1997
Start Page
154
End Page
164
DOI
10.1006/jmbi.1997.1031

The energy landscape of a fast-folding protein mapped by Ala-->Gly substitutions.

A moderately stable protein with typical folding kinetics unfolds and refolds many times during its cellular lifetime. In monomeric lambda repressor this process is extremely rapid, with an average folded state lifetime of only 30 milliseconds. A thermostable variant of this protein (G46A/G48A) unfolds with the wild-type rate, but it folds in approximately 20 microseconds making it the fastest-folding protein yet observed. The effects of alanine to glycine substitutions on the folding and unfolding rate constants of the G46A/G48A variant, measured by dynamic NMR spectroscopy, indicate that the transition state is an ensemble comprised of a disperse range of conformations. This structural diversity in the transition state is consistent with the idea that folding chains are directed towards the native state by a smooth funnel-like conformational energy landscape. The kinetic data for the folding of monomeric lambda repressor can be understood by merging the new energy landscape view of folding with traditional models. This hybrid model incorporates the conformational diversity of denatured and transition state ensembles, a transition state activation energy, and the importance of intrinsic helical stabilities.

Authors
Burton, RE; Huang, GS; Daugherty, MA; Calderone, TL; Oas, TG
MLA Citation
Burton, RE, Huang, GS, Daugherty, MA, Calderone, TL, and Oas, TG. "The energy landscape of a fast-folding protein mapped by Ala-->Gly substitutions." Nat Struct Biol 4.4 (April 1997): 305-310.
PMID
9095199
Source
pubmed
Published In
Nature Structural Biology
Volume
4
Issue
4
Publish Date
1997
Start Page
305
End Page
310

Microsecond protein folding through a compact transition state.

Dynamic NMR methods have been employed to measure the folding and unfolding rate constants of two extremely fast-folding proteins. lambda 6-85, a truncated, monomeric form of the N-terminal domain of lambda repressor, refolds with a lifetime of approximately 250 microseconds. These methods have also been applied to a thermostable lambda 6-85 variant with alanine substituted for glycine residues 46 and 48 in the third helix (G46A/G48A). Both proteins exhibit linear ln (kf,u) versus [urea] plots, consistent with two-state folding for both proteins. When extrapolated to 0M urea, the data indicate that G46A/G48A folds with a lifetime of less than 20 microseconds. The slopes of the ln (kf,u) versus [urea] curves (mu and mf) indicate that the modest Gly-->Ala double mutation dramatically changes the transition state solvent accessibility. The transition state for lambda 6-85 has a fractional accessibility (mu/(mu-mf)) of 0.61, whereas the transition state for G46A/G48A is much more native-like, with a fractional accessibility of 0.16. The extraordinary change in the folding pathway that these mutations induce suggests that the intrinsic stability of helix 3 is an important determinant of the folding mechanism.

Authors
Burton, RE; Huang, GS; Daugherty, MA; Fullbright, PW; Oas, TG
MLA Citation
Burton, RE, Huang, GS, Daugherty, MA, Fullbright, PW, and Oas, TG. "Microsecond protein folding through a compact transition state." J Mol Biol 263.2 (October 25, 1996): 311-322.
PMID
8913309
Source
pubmed
Published In
Journal of Molecular Biology
Volume
263
Issue
2
Publish Date
1996
Start Page
311
End Page
322
DOI
10.1006/jmbi.1996.0577

High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli.

The expression of eukaryotic genes in Escherichia coli is one of the most frequently used tools of modern science. The arginine codon AGA is a common codon in eukaryotic genes but is particularly rare in E. coli. We report here 36 to 42% misincorporation of lysine at three AGA codons in a well-expressed protein. This misincorporation yields a protein whose electrospray mass spectrum (ESMS) shows peaks at the expected mass (M), M-28, M-56 and M-84 with intensities representing 34.5(+/-0.7), 37.5(+/-1.1), 21.2(+/-1.7) and 6.6(+/-0.5) % of the total intensity, respectively. Replacement of either all three AGA codons or the two closest to the 3' end of the gene by the more common CGC arginine codon gave a protein with a single ESMS peak. Misincorporation could also be eliminated by the co-expression of the tRNA(UCL)Arg gene, argU. These studies demonstrate that misincorporation of amino acids at rare codons of recombinant proteins can be far higher than previously thought.

Authors
Calderone, TL; Stevens, RD; Oas, TG
MLA Citation
Calderone, TL, Stevens, RD, and Oas, TG. "High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli." J Mol Biol 262.4 (October 4, 1996): 407-412.
PMID
8893852
Source
pubmed
Published In
Journal of Molecular Biology
Volume
262
Issue
4
Publish Date
1996
Start Page
407
End Page
412
DOI
10.1006/jmbi.1996.0524

Heat and cold denatured states of monomeric lambda repressor are thermodynamically and conformationally equivalent.

Although the denaturation of proteins by low temperatures is a well-documented phenomenon, little is known about the molecular details of the process. In this study, the parameters describing the denaturation thermodynamics of residues 6-85 of the N-terminal domain of lambda repressor have been determined by fitting the three-dimensional thermal-urea denaturation surface obtained by circular dichroism. The shape of the surface shows cold denaturation at low temperatures and urea concentrations above 2 M, which allows accurate determination of the apparent heat capacity of denaturation (delta Cp). Denaturation curves based on aromatic 1H NMR spectra give identical denaturation curves, confirming purely twostate folding under all conditions studies. The denaturation surface can be fit with constant delta Cp and delta In KD/delta[urea] (KD is the equilibrium constant for denaturation), consistent with a thermodynamically invariant denatured state. In addition, the aromatic 1H NMR spectrum of the cold denatured state at 0 degree C in 3 M uea is essentially identical to the spectrum at 70 degree C in 3 M urea. These observations indicate that the structures of the cold and heat denatured states, in the presence of 3 M urea, are thermodynamically and conformationally equivalent.

Authors
Huang, GS; Oas, TG
MLA Citation
Huang, GS, and Oas, TG. "Heat and cold denatured states of monomeric lambda repressor are thermodynamically and conformationally equivalent." Biochemistry 35.20 (May 21, 1996): 6173-6180.
PMID
8639557
Source
pubmed
Published In
Biochemistry
Volume
35
Issue
20
Publish Date
1996
Start Page
6173
End Page
6180
DOI
10.1021/bi960250l

Heat and cold denatured states of monomeric λ represser are thermodynamically and conformationally equivalen

Although the denaturation of proteins by low temperatures is a well-documented phenomenon, little is known about the molecular details of the process. In this study, the parameters describing the denaturation thermodynamics of residues 6-85 of the N-terminal domain of λ repressor have been determined by fitting the three-dimensional thermal-urea denaturation surface obtained by circular dichroism. The shape of the surface shows cold denaturation at low temperatures and urea concentrations above 2 M, which allows accurate determination of the apparent heat capacity of denaturation (ΔCp). Denaturation curves based on aromatic 1H NMR spectra give identical denaturation curves, confirming purely two-state folding under all conditions studied. The denaturation surface can be fit with constant ΔCP and δ In KD/δ[urea] (KD is the equilibrium constant for denaturation), consistent with a thermodynamically invariant denatured state. In addition, the aromatic 1H NMR spectrum of the cold denatured state at 0 °C in 3 M urea is essentially identical to the spectrum at 70 °C in 3 M urea. These observations indicate that the structures of the cold and heat denatured states, in the presence of 3 M urea, are thermodynamically and conformationally equivalent.

Authors
Huang, GS; Oas, TG
MLA Citation
Huang, GS, and Oas, TG. "Heat and cold denatured states of monomeric λ represser are thermodynamically and conformationally equivalen." Biochemistry 35.20 (1996): X-6180.
Source
scival
Published In
Biochemistry
Volume
35
Issue
20
Publish Date
1996
Start Page
X
End Page
6180

Submillisecond folding of monomeric lambda repressor.

The folding kinetics of a truncated form of the N-terminal domain of phage lambda repressor [lambda 6-85] has been investigated by using the technique of dynamic NMR. lambda 6-85 has been shown previously to fold in a purely two-state fashion. This allows the determination of folding and unfolding rates from simulation of the exchange-broadened aromatic resonances of Tyr-22. The folding kinetics were determined over a range of 1.35 to 3.14 M urea. The urea dependence of both folding and unfolding rate constants is exponential, suggesting that the rate-determining step is invariant at the urea concentrations studied. The folding and unfolding rates extrapolated to 0 M urea at 37 degrees C are 3600 +/- 400 s-1 and 27 +/- 6 s-1, respectively. The observed lambda 6-85 folding rate constant exceeds that of other fast-folding globular proteins by a factor of 14-54. The urea dependence of the folding and unfolding rate constants suggests that the transition state of the rate-determining step is considerably more exposed to solvent than previously studied protein-folding transition states. The surprising rapidity of lambda 6-85 folding and unfolding may be the consequence of its all-helical secondary structure. These kinetic results clearly demonstrate that all of the fundamental events of protein folding can occur on the submillisecond time scale.

Authors
Huang, GS; Oas, TG
MLA Citation
Huang, GS, and Oas, TG. "Submillisecond folding of monomeric lambda repressor." Proc Natl Acad Sci U S A 92.15 (July 18, 1995): 6878-6882.
PMID
7624336
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
92
Issue
15
Publish Date
1995
Start Page
6878
End Page
6882

Structure and stability of monomeric lambda repressor: NMR evidence for two-state folding.

The absence of equilibrium intermediates in protein folding reactions (i.e., two-state folding) simplifies thermodynamic and kinetic analyses but is difficult to prove rigorously. We demonstrate a sensitive method for detecting partially folded species based on using proton chemical shifts as local probes of structure. The coincidence of denaturation curves for probes throughout the molecule is a particularly stringent test for two-state folding. In this study we investigate a new form of the N-terminal domain of bacteriophage lambda repressor consisting of residues 6-85 (lambda 6-85) using nuclear magnetic resonance (NMR) and circular dichroism (CD). This truncated version lacks the residues required for dimerization and is monomeric under the conditions used for NMR. Heteronuclear NMR was used to assign the 1H, 15N, and backbone 13C resonances. The secondary and tertiary structure of lambda 6-85 is very similar to that reported for the crystal structure of the DNA-bound 1-92 fragment [Beamer, L. J., and Pabo, C. O. (1992) J. Mol. Biol. 227, 177-196], as judged by analysis of chemical shifts, amide hydrogen exchange, amide-alpha coupling constants, and nuclear Overhauser enhancements. Thermal and urea denaturation studies were conducted using the chemical shifts of the four aromatic side chains as local probes and the CD signal at 222 nm as a global probe. Plots of the fraction denatured versus denaturant concentration obtained from these studies are identical for all probes under all conditions studied. This observation provides strong evidence for two-state folding, indicating that there are no populated intermediates in the folding of lambda 6-85.

Authors
Huang, GS; Oas, TG
MLA Citation
Huang, GS, and Oas, TG. "Structure and stability of monomeric lambda repressor: NMR evidence for two-state folding." Biochemistry 34.12 (March 28, 1995): 3884-3892.
PMID
7696251
Source
pubmed
Published In
Biochemistry
Volume
34
Issue
12
Publish Date
1995
Start Page
3884
End Page
3892

An anionic diplatinum DNA photocleavage agent: Chemical mechanism and footprinting of λ repressor

The dσ* → pσ excited state of Pt2(pop)44- (1, pop = P2O5H22-) elicits frank scission of double-stranded DNA as assayed by high-resolution gel electrophoresis. The photoreaction of 1 and a 5'-32P-labeled 25-mer duplex produces a surprisingly even ladder of phosphate terminated bands with some modified bands that can be assigned as phosphoglycolate termini by comigration with the products of an Fe(EDTA)2-/H2O2 reaction. The analogous reaction of the 3'-32P-labeled duplex also produces phosphate termini and a modified band that can be assigned as a 5'-aldehyde terminus by NaBH4 reduction to the 5'-alcohol and comigration with authentic alcohol termini generated using alkaline phosphatase. These products are consistent with abstraction of the 4' and 5' hydrogens from the deoxyribose function; products indicative of 1' or 3' chemistry were not detected. The reaction is more efficient in the presence of O2, which appears to trap the radical produced by homolytic C-H activation. The even cleavage ladder argues strongly against a 1O2 mechanism, and the cleavage is not enhanced in D2O. Further, ethanol does not inhibit the reaction of 1 at concentrations up to 1 M, where the reaction of hydroxyl radical is completely quenched. These experiments point to a mechanism where the tetraanionic complex collides directly with the DNA to effect C-H activation, which is supported by a strong enhancement in cleavage by Mg2+. This unusual reaction has been used to obtain a footprint of λ repressor bound to the O(R)1 sequence. The resolution of the footprint is similar to that of hydroxyl radical, which permits binding of the repressor to a single side of the DNA helix to be distinguished.

Authors
Breiner, KM; Daugherty, MA; Oas, TG; Thorp, HH
MLA Citation
Breiner, KM, Daugherty, MA, Oas, TG, and Thorp, HH. "An anionic diplatinum DNA photocleavage agent: Chemical mechanism and footprinting of λ repressor." Journal of the American Chemical Society 117.47 (1995): 11673-11679.
Source
scival
Published In
Journal of the American Chemical Society
Volume
117
Issue
47
Publish Date
1995
Start Page
11673
End Page
11679
DOI
10.1021/ja00152a007

Propensity for a leucine zipper-like domain of human immunodeficiency virus type 1 gp41 to form oligomers correlates with a role in virus-induced fusion rather than assembly of the glycoprotein complex.

For a number of viruses, oligomerization is a critical component of envelope processing and surface expression. Previously, we reported that a synthetic peptide (DP-107) corresponding to the putative leucine zipper region (aa 553-590) of the transmembrane protein (gp41) of human immunodeficiency virus type 1 (HIV-1) exhibited alpha-helical secondary structure and self-associated as a coiled coil. In view of the tendency of this type of structure to mediate protein association, we speculated that this region of gp41 might play a role in HIV-1 envelope oligomerization. However, later it was shown that mutations which should disrupt the structural elements of this region of gp41 did not affect envelope processing, transport, or surface expression (assembly oligomerization). In this report we compare the effects of amino acid substitutions within this coiled-coil region on structure and function of both viral envelope proteins and the corresponding synthetic peptides. Our results establish a correlation between the destabilizing effects of amino acid substitutions on coiled-coil structure in the peptide model and phenotype of virus entry. These biological and physical biochemical studies do not support a role for the coiled-coil structure in mediating the assembly oligomerization of HIV-1 envelope but do imply that this region of gp41 plays a key role in the sequence of events associated with viral entry. We propose a functional role for the coiled-coil domain of HIV-1 gp41.

Authors
Wild, C; Dubay, JW; Greenwell, T; Baird, T; Oas, TG; McDanal, C; Hunter, E; Matthews, T
MLA Citation
Wild, C, Dubay, JW, Greenwell, T, Baird, T, Oas, TG, McDanal, C, Hunter, E, and Matthews, T. "Propensity for a leucine zipper-like domain of human immunodeficiency virus type 1 gp41 to form oligomers correlates with a role in virus-induced fusion rather than assembly of the glycoprotein complex." Proc Natl Acad Sci U S A 91.26 (December 20, 1994): 12676-12680.
PMID
7809100
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
91
Issue
26
Publish Date
1994
Start Page
12676
End Page
12680

Springs and hinges: dynamic coiled coils and discontinuities.

Authors
Oas, TG; Endow, SA
MLA Citation
Oas, TG, and Endow, SA. "Springs and hinges: dynamic coiled coils and discontinuities." Trends Biochem Sci 19.2 (February 1994): 51-54. (Review)
PMID
8160263
Source
pubmed
Published In
Trends in Biochemical Sciences
Volume
19
Issue
2
Publish Date
1994
Start Page
51
End Page
54
DOI
10.1016/0968-0004(94)90030-2

STRUCTURE AND ENERGETICS OF CONCANAVALIN A-OLIGOSACCHARIDE BINDING

Authors
CHERVENAK, MC; OAS, TG; TOONE, EJ
MLA Citation
CHERVENAK, MC, OAS, TG, and TOONE, EJ. "STRUCTURE AND ENERGETICS OF CONCANAVALIN A-OLIGOSACCHARIDE BINDING." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 204 (August 23, 1992): 102-CARB.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
204
Publish Date
1992
Start Page
102
End Page
CARB

A synthetic peptide inhibitor of human immunodeficiency virus replication: Correlation between solution structure and viral inhibition

A peptide designated DP-107 was synthesized containing amino acid residues 558-595 of the envelope glycoprotein gp160 of human immunodeficiency virus type 1 strain LAI (HIV-1LAI)- Algorithms for secondary structure have predicted that this region of the envelope transmembrane protein should form an extended α-helix. Consistent with this prediction, analysis by circular dichroism (CD) indicated that, under physiological conditions, DP-107 is ≈85% helical. The high degree of stable secondary structure in a synthetic peptide of this size suggests self-association typical of a coiled coil or leucine zipper. In biological assays, the peptide efficiently blocked virus-mediated cell-cell fusion processes as well as infection of peripheral blood mononuclear cells by both prototypic and primary isolates of HIV-1. A single amino acid substitution in the peptide greatly destabilized its solution structure as measured by CD and abrogated its antiviral activity. An analogue containing a terminal cysteine was oxidized to form a dimer, and this modification lowered the dose required for antiviral effect from 5 to about 1 μg/ml. These results suggest that both oligomerization and ordered structure are necessary for biological activity. They provide insights also into the role of this region in HIV infection and the potential for development of a new class of antiviral agents.

Authors
Wild, C; Oas, T; Mcdanal, C; Bolognesi, D; Matthews, T
MLA Citation
Wild, C, Oas, T, Mcdanal, C, Bolognesi, D, and Matthews, T. "A synthetic peptide inhibitor of human immunodeficiency virus replication: Correlation between solution structure and viral inhibition." Proceedings of the National Academy of Sciences of the United States of America 89.21 (1992): 10537-10541.
PMID
1438243
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
89
Issue
21
Publish Date
1992
Start Page
10537
End Page
10541
DOI
10.1073/pnas.89.21.10537

Inhibition of thermolysin by phosphonamidate transition-state analogues: measurement of 31P-15N bond lengths and chemical shifts in two enzyme-inhibitor complexes by solid-state nuclear magnetic resonance.

31P and 15N chemical shifts and 31P-15N bond lengths have been measured with solid-state NMR techniques in two inhibitors of thermolysin, carbobenzoxy-Glyp-L-Leu-L-Ala (ZGpLA) and carbobenzoxy-L-Phep-L-Leu-L-Ala (ZFpLA), both as free lithium salts and when bound to the enzyme. Binding of both inhibitors to thermolysin results in large changes in the 31P chemical shifts. These changes are more dramatic for the tighter binding inhibitor ZFpLA, where a approximately 20 ppm downfield movement of the 31P isotropic chemical shift (sigma iso) is observed. This shift is due to changes in the shift tensor elements sigma 11 and sigma 22, while sigma 33 remains essentially constant. We observed a similar pattern for ZGpLA, but only a approximately 5 ppm change occurs in sigma iso. The changes in the 15N chemical shifts for both inhibitors are small upon binding, amounting to downfield shifts of 2 and 4 ppm for ZGpLA and ZFpLA, respectively. This indicates that there are no changes in the protonation state of the 15N in either the ZFpLA- or the ZGpLA-thermolysin complex. NMR distance measurements yield a P-N bond length rP-N = 1.68 +/- 0.03 A for the tight binding inhibitor ZFpLA both in its free lithium salt form and in its thermolysin-ZFpLA complex, a distance that is much shorter than the 1.90-A distance reported by X-ray crystallography studies [Holden et al. (1987) Biochemistry 26, 8542-8553].(ABSTRACT TRUNCATED AT 250 WORDS)

Authors
Copié, V; Kolbert, AC; Drewry, DH; Bartlett, PA; Oas, TG; Griffin, RG
MLA Citation
Copié, V, Kolbert, AC, Drewry, DH, Bartlett, PA, Oas, TG, and Griffin, RG. "Inhibition of thermolysin by phosphonamidate transition-state analogues: measurement of 31P-15N bond lengths and chemical shifts in two enzyme-inhibitor complexes by solid-state nuclear magnetic resonance." Biochemistry 29.39 (October 2, 1990): 9176-9184.
PMID
2271586
Source
pubmed
Published In
Biochemistry
Volume
29
Issue
39
Publish Date
1990
Start Page
9176
End Page
9184

Secondary structure of a leucine zipper determined by nuclear magnetic resonance spectroscopy.

Previous work has shown that a synthetic peptide corresponding to the leucine zipper region of the yeast transcriptional activator GCN4 forms a stable dimer of alpha-helices and that the helices are oriented in a parallel manner. Two-dimensional nuclear magnetic resonance spectroscopy (NMR) is used here to demonstrate that the helix is continuous for at least 32 of the 33 residues in the peptide. The results also indicate that the dimer is symmetric. It is therefore unlikely that the interdigitation model for the structure of leucine zippers is correct, since interdigitation of leucine residues in a parallel dimer would lead to an asymmetric structure. The data are consistent with a coiled-coil structure.

Authors
Oas, TG; McIntosh, LP; O'Shea, EK; Dahlquist, FW; Kim, PS
MLA Citation
Oas, TG, McIntosh, LP, O'Shea, EK, Dahlquist, FW, and Kim, PS. "Secondary structure of a leucine zipper determined by nuclear magnetic resonance spectroscopy." Biochemistry 29.12 (March 27, 1990): 2891-2894.
PMID
2337572
Source
pubmed
Published In
Biochemistry
Volume
29
Issue
12
Publish Date
1990
Start Page
2891
End Page
2894

PEPTIDE MODELS AND THE FOLDING OF BPTI

Authors
OAS, TG; STALEY, JP; KWON, DY; GOODMAN, EM; RUKOWSKI, R; KIM, PS
MLA Citation
OAS, TG, STALEY, JP, KWON, DY, GOODMAN, EM, RUKOWSKI, R, and KIM, PS. "PEPTIDE MODELS AND THE FOLDING OF BPTI." BIOPHYSICAL JOURNAL 57.2 (February 1990): A3-A3.
Source
wos-lite
Published In
Biophysical Journal
Volume
57
Issue
2
Publish Date
1990
Start Page
A3
End Page
A3

Peptides from Conus venoms which affect Ca++ entry into neurons

The venoms of fish-hunting Conus contain paralytic conotoxins and an unprecedented variety of other biologically-active peptides. Particularly noteworthy are peptides which inhibit calcium entry into neurons, the conantokins and ω-conotoxins which target NMDA receptors and voltage-sensitive Ca channels respectively. The conantokins contain at least four residues of γ-carboxyglutamate (Gla), a post-translationally modified amino acid. Conantokins are folded into an α-helical conformation and bind acidic membranes in a calcium-dependent manner. Upon envenomation, conantokins may concentrate on appropriate membranes and target to peripheral fish NMDA receptors located on such membranes. The ω-conotoxins are disulfide bonded peptides; a comparison of several ω-conotoxin sequences reveals considerable sequence variability. Recent studies with ω-conotoxins have also revealed a developmental specificity in mice. The results suggest that elements controlling breathing in neonatal mammals, but not in adults, are ω-conotoxin sensitive.

Authors
Myers, RA; McIntosh, JM; Imperial, J; Williams, RW; Oas, T; Haack, JA; Hernandez, J-F; Rivier, J; Cruz, LJ; Olivera, BM
MLA Citation
Myers, RA, McIntosh, JM, Imperial, J, Williams, RW, Oas, T, Haack, JA, Hernandez, J-F, Rivier, J, Cruz, LJ, and Olivera, BM. "Peptides from Conus venoms which affect Ca++ entry into neurons." Journal of Toxicology - Toxin Reviews 9.2 (1990): 179-202.
Source
scival
Published In
Toxin Reviews
Volume
9
Issue
2
Publish Date
1990
Start Page
179
End Page
202

A PROTEIN FOLDING INTERMEDIATE ANALOG - DESIGN, SYNTHESIS, AND CHARACTERIZATION

Authors
OAS, TG; KIM, PS
MLA Citation
OAS, TG, and KIM, PS. "A PROTEIN FOLDING INTERMEDIATE ANALOG - DESIGN, SYNTHESIS, AND CHARACTERIZATION." 1990.
Source
wos-lite
Published In
PROTEIN FOLDING : DECIPHERING THE SECOND HALF OF THE GENETIC CODE
Publish Date
1990
Start Page
123
End Page
128

SELECTIVE NMR DETECTION OF C-13-N-15 DIPOLE PAIRS IN SOLID SAMPLES

Authors
OAS, TG; HARTZELL, CJ; DROBNY, GP; DAHLQUIST, FW
MLA Citation
OAS, TG, HARTZELL, CJ, DROBNY, GP, and DAHLQUIST, FW. "SELECTIVE NMR DETECTION OF C-13-N-15 DIPOLE PAIRS IN SOLID SAMPLES." JOURNAL OF MAGNETIC RESONANCE 81.2 (February 1, 1989): 395-399.
Source
wos-lite
Published In
Journal of Magnetic Resonance (1969)
Volume
81
Issue
2
Publish Date
1989
Start Page
395
End Page
399
DOI
10.1016/0022-2364(89)90072-3

Two-Dimensional Hybrid Experiments for the Measurement of Small Anisotropies in Magic-Angle Spinning Nuclear Magnetic Resonance

Authors
Kolbert, AC; De Groot, HJM; Oas, TG; Griffin, RG
MLA Citation
Kolbert, AC, De Groot, HJM, Oas, TG, and Griffin, RG. "Two-Dimensional Hybrid Experiments for the Measurement of Small Anisotropies in Magic-Angle Spinning Nuclear Magnetic Resonance." Advances in Magnetic and Optical Resonance 13.C (January 1, 1989): 183-194.
Source
scopus
Published In
Advances in Magnetic and Optical Resonance
Volume
13
Issue
C
Publish Date
1989
Start Page
183
End Page
194
DOI
10.1016/B978-0-12-025513-9.50013-6

Selective NMR detection of 13C15N dipole pairs in solid samples

Authors
Oas, TG; Hartzell, CJ; Drobny, GP; Dahlquist, FW
MLA Citation
Oas, TG, Hartzell, CJ, Drobny, GP, and Dahlquist, FW. "Selective NMR detection of 13C15N dipole pairs in solid samples." Journal of Magnetic Resonance (1969) 81.2 (1989): 395-399.
Source
scival
Published In
Journal of Magnetic Resonance (1969)
Volume
81
Issue
2
Publish Date
1989
Start Page
395
End Page
399

2-DIMENSIONAL HYBRID EXPERIMENTS FOR THE MEASUREMENT OF SMALL ANISOTROPIES IN MAGIC-ANGLE SPINNING NUCLEAR-MAGNETIC-RESONANCE

Authors
KOLBERT, AC; DEGROOT, HJM; OAS, TG; GRIFFIN, RG
MLA Citation
KOLBERT, AC, DEGROOT, HJM, OAS, TG, and GRIFFIN, RG. "2-DIMENSIONAL HYBRID EXPERIMENTS FOR THE MEASUREMENT OF SMALL ANISOTROPIES IN MAGIC-ANGLE SPINNING NUCLEAR-MAGNETIC-RESONANCE." 1989.
Source
wos-lite
Published In
ADVANCES IN MAGNETIC RESONANCE, VOL 13
Volume
13
Publish Date
1989
Start Page
183
End Page
194

A peptide model of a protein folding intermediate.

It is difficult to determine the structures of protein folding intermediates because folding is a highly cooperative process. A disulphide-bonded peptide pair, designed to mimic the first crucial intermediate in the folding of bovine pancreatic trypsin inhibitor, contains secondary and tertiary structure similar to that found in the native protein. Peptide models like this circumvent the problem of cooperativity and permit characterization of structures of folding intermediates.

Authors
Oas, TG; Kim, PS
MLA Citation
Oas, TG, and Kim, PS. "A peptide model of a protein folding intermediate." Nature 336.6194 (November 3, 1988): 42-48.
PMID
3185721
Source
pubmed
Published In
Nature
Volume
336
Issue
6194
Publish Date
1988
Start Page
42
End Page
48
DOI
10.1038/336042a0

A new iterative least-squares method for the extraction of NMR parameters from nonideal powder patterns

A new approach to fitting powder patterns via nonlinear least-squares analysis is described. This fitting method is designed to be particularly sensitive to the frequencies of important features and insensitive to discrepancies in intensity between calculated and observed spectra. A description of an optimized powder pattern calculation routine is given with a discussion of the calculation of chemical-shift, dipole-coupled chemical-shift, and nonaxially symmetric quadrupolar powder spectra. Examples of fits of chemical-shift and dipole-coupled chemical-shift powder patterns are shown. The potential applications and limitations of the technique are discussed. © 1988.

Authors
Oas, TG; Drobny, GP; Dahlquistt, FW
MLA Citation
Oas, TG, Drobny, GP, and Dahlquistt, FW. "A new iterative least-squares method for the extraction of NMR parameters from nonideal powder patterns." Journal of Magnetic Resonance (1969) 78.3 (1988): 408-424.
Source
scival
Published In
Journal of Magnetic Resonance (1969)
Volume
78
Issue
3
Publish Date
1988
Start Page
408
End Page
424

Enhancement of the effect of small anisotropies in magic-angle spinning nuclear magnetic resonance

A variety of novel methods in magic-angle spinning NMR is described. The effects have in common the enhancement of the influence of small anisotropies on the NMR spectrum by deliberate intervention, i.e. either by applying pulses, carefully chosen continuous r.f. fields, or by adjustment of the spinning speed. It is shown that rotational sidebands in two-dimensional spin-echo NMR are often much larger than in one-dimensional NMR, owing to the interference of the π-pulse with the rotational echo formation. It is also demonstrated that in systems containing heteronuclear spin pairs the application of a weak continuous r.f. field of carefully chosen intensity can reintroduce small heteronuclear couplings into the spectrum. This is known as rotational resonance recoupling and is due to the interference of coherent spin rotations with the normal averaging effect of the sample rotation. Related effects can occur in homonuclear spin systems when an integer multiple of the spinning speed matches the difference between isotropic chemical shifts. In this case no extra r.f. field is necessary to amplify the effect of the non-secular parts of the dipolar interaction. Greatly enhanced polarization exchange as well as strong spectral effects are demonstrated. The application of these novel methods to the measurement of small interaction tensors and thereby to the extraction of important structural information is discussed.

Authors
Raleigh, DP; Kolbert, AC; Oas, TG; Levitt, MH; Griffin, RG
MLA Citation
Raleigh, DP, Kolbert, AC, Oas, TG, Levitt, MH, and Griffin, RG. "Enhancement of the effect of small anisotropies in magic-angle spinning nuclear magnetic resonance." Journal of the Chemical Society, Faraday Transactions 1: Physical Chemistry in Condensed Phases 84.11 (1988): 3691-3711.
Source
scival
Published In
Journal of the Chemical Society, Faraday Transactions I
Volume
84
Issue
11
Publish Date
1988
Start Page
3691
End Page
3711
DOI
10.1039/F19888403691

Rotary resonance recoupling of dipolar interactions in solid-state nuclear magnetic resonance spectroscopy

A new resonance effect in solid-state nuclear magnetic resonance (NMR) is described. The effect involves a combination of magic-angle sample rotation with irradiation of a heteronuclear spin system at the Larmor frequency of one of the spin species. If the irradiation intensity is such as to establish a match between spin nutation and sample rotation, it is shown that the heteronuclear dipolar spin interaction is selectively reintroduced into the spectrum. This allows small dipolar coupling constants to be measured in the presence of large shielding anisotropies. Applications are anticipated for determination of internuclear distances in materials lacking long-range order, such as polycrystalline materials, polymers, and surfaces. © 1988 American Institute of Physics.

Authors
Oas, TG; Griffin, RG; Levitt, MH
MLA Citation
Oas, TG, Griffin, RG, and Levitt, MH. "Rotary resonance recoupling of dipolar interactions in solid-state nuclear magnetic resonance spectroscopy." The Journal of Chemical Physics 89.2 (1988): 692-695.
Source
scival
Published In
Journal of Chemical Physics
Volume
89
Issue
2
Publish Date
1988
Start Page
692
End Page
695

ROTARY RESONANCE RECOUPLING IN HETERONUCLEAR SPIN PAIR SYSTEMS

Authors
LEVITT, MH; OAS, TG; GRIFFIN, RG
MLA Citation
LEVITT, MH, OAS, TG, and GRIFFIN, RG. "ROTARY RESONANCE RECOUPLING IN HETERONUCLEAR SPIN PAIR SYSTEMS." ISRAEL JOURNAL OF CHEMISTRY 28.4 (1988): 271-282.
Source
wos-lite
Published In
Israel Journal of Chemistry
Volume
28
Issue
4
Publish Date
1988
Start Page
271
End Page
282

DETERMINATION OF THE N-15 AND C-13 CHEMICAL-SHIFT TENSORS OF L-[C-13]ALANYL-L-[N-15]ALANINE FROM THE DIPOLE-COUPLED POWDER PATTERNS

Authors
HARTZELL, CJ; WHITFIELD, M; OAS, TG; DROBNY, GP
MLA Citation
HARTZELL, CJ, WHITFIELD, M, OAS, TG, and DROBNY, GP. "DETERMINATION OF THE N-15 AND C-13 CHEMICAL-SHIFT TENSORS OF L-[C-13]ALANYL-L-[N-15]ALANINE FROM THE DIPOLE-COUPLED POWDER PATTERNS." JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 109.20 (September 30, 1987): 5966-5969.
Source
wos-lite
Published In
Journal of the American Chemical Society
Volume
109
Issue
20
Publish Date
1987
Start Page
5966
End Page
5969
DOI
10.1021/ja00254a012

A NEW APPROACH TO LEAST-SQUARES FITTING ANALYSIS OF NMR POWDER PATTERNS

Authors
OAS, TG; HARTZELL, CJ; DAHLQUIST, FW; DROBNY, GP
MLA Citation
OAS, TG, HARTZELL, CJ, DAHLQUIST, FW, and DROBNY, GP. "A NEW APPROACH TO LEAST-SQUARES FITTING ANALYSIS OF NMR POWDER PATTERNS." BIOPHYSICAL JOURNAL 51.2 (February 1987): A80-A80.
Source
wos-lite
Published In
Biophysical Journal
Volume
51
Issue
2
Publish Date
1987
Start Page
A80
End Page
A80

The amide 15N chemical shift tensors of four peptides determined from 13C dipole-coupled chemical shift powder patterns

The 15N chemical shift tensors of a homologous series of peptides of the form N-acetyl[1-13C]glycyl[15N]-X-amide (X = glycine, alanine, and tyrosine) and the unprotected dipeptide [1-13C]glycyl[15N]glycine hydrochloride have been determined from 13C dipole-coupled 15N powder patterns. It was found that the shift tensor principal values differ greatly while their molecular orientation does not. The common shift tensor orientation places σ22 perpendicular to the peptide plane, and σ33 at a 99° angle with respect to the C-N bond. The orientations of σ11 and σ22 were previously unknown for 15N chemical shift tensors of amides. Comparison of magic angle spinning (MAS) spectra with solution spectra shows significantly different solid and solution isotropic chemical shifts for several of the peptides studied, demonstrating that at least part of the variation in principal values is due to lattice effects. This conclusion is borne out by the MAS spectrum of N-acetyl[1-13C]glycl-[15N]phenylalaninamide, which shows at least three peaks corresponding to different lattice environments. © 1987 American Chemical Society.

Authors
Oas, TG; Hartzell, CJ; Dahlquist, FW; Drobny, GP
MLA Citation
Oas, TG, Hartzell, CJ, Dahlquist, FW, and Drobny, GP. "The amide 15N chemical shift tensors of four peptides determined from 13C dipole-coupled chemical shift powder patterns." Journal of the American Chemical Society 109.20 (1987): 5962-5966.
Source
scival
Published In
Journal of the American Chemical Society
Volume
109
Issue
20
Publish Date
1987
Start Page
5962
End Page
5966

Determination of the 15N and 13C chemical shift tensors of L-[13C]alanyl-L-[15N]alanine from the dipole-coupled powder patterns

The 13C and 15N chemical shift tensors of L-[1-13C]alanyl-L-[15N]alanine have been determined from the dipole-coupled powder patterns and verified with the decoupled spectra. The principal values of the 13C tensor are σ11 = -115.5, σ22 = -42.3, and σ33 = 33.5 ppm, and the polar angles relating the 13C-15N bond to the chemical shift tensor are β = 90° and α = -39.5°. Although the 13C powder pattern widths of AlaAla and AcGlyAlaNH2 differ by only 2%, the anisotropy and asymmetry parameters differ by 10 and 25%, respectively. The principal values for the 15N chemical shift tensor are σ11= 65.3, σ22 = 78.1, and σ33 = 215.5 ppm, and the orientation of the 13C-15N bond to the σ33 axis is 106°. AlaAla and AcGlyAlaNH2 show a striking difference in asymmetry parameters (0.06 vs. 0.16) and anisotropy (144 vs. 165). © 1987 American Chemical Society.

Authors
Hartzell, CJ; Whitfield, M; Oas, TG; Drobny, GP
MLA Citation
Hartzell, CJ, Whitfield, M, Oas, TG, and Drobny, GP. "Determination of the 15N and 13C chemical shift tensors of L-[13C]alanyl-L-[15N]alanine from the dipole-coupled powder patterns." Journal of the American Chemical Society 109.20 (1987): 5966-5969.
Source
scival
Published In
Journal of the American Chemical Society
Volume
109
Issue
20
Publish Date
1987
Start Page
5966
End Page
5969

The carbonyl 13C chemical shift tensors of five peptides determined from 15N dipole-coupled chemical shift powder patterns

The 13C chemical shift tensors have been determined for the glycine carbonyl carbon in a homologous series of peptides of the general form N-acetyl[1-13C]glycyl-X-amide, where X was [15N]glycine, DL-[15N]-tyrosine, L-[15N]phenylalanine and DL-[15N]alanine. The principal values and molecular orientations of the tensors were extracted from 15N dipole-coupled 13C powder spectra. The shift tensor of a powdered sample of [1-13C]glycyl[15N]glycine·HCl·H2O was determined by the same method and was found to agree to within 1 ppm in principal values and 2° in orientation with the previous single crystal measurements of R. E. Stark et al. (J. Magn. Reson. 1983, 55, 266). The shift tensors of the five peptides were found to be significantly different in both principal values and molecular orientation. However, the isotropic chemical shifts of the end-protected peptides in D2O were nearly identical. From these data it is concluded that lattice environment has a significant effect on the chemical shift tensors of peptide carbonyl carbons. An approach to approximating carbonyl 13C chemical shift tensors of peptides in proteins with use of the isotropic chemical shift in the molecule of interest is proposed. In addition, the utility of the powder pattern technique for accurately determining the chemical shift tensors of peptide carbonyl carbons is demonstrated. © 1987 American Chemical Society.

Authors
Oas, TG; Hartzell, CJ; McMahon, TJ; Drobny, GP; Dahlquist, FW
MLA Citation
Oas, TG, Hartzell, CJ, McMahon, TJ, Drobny, GP, and Dahlquist, FW. "The carbonyl 13C chemical shift tensors of five peptides determined from 15N dipole-coupled chemical shift powder patterns." Journal of the American Chemical Society 109.20 (1987): 5956-5962.
Source
scival
Published In
Journal of the American Chemical Society
Volume
109
Issue
20
Publish Date
1987
Start Page
5956
End Page
5962

N-15 AMIDE CHEMICAL-SHIFT TENSORS OF SEVERAL DIPEPTIDES

Authors
OAS, TG; HARTZELL, CJ; DAHLQUIST, FW; DROBNY, G
MLA Citation
OAS, TG, HARTZELL, CJ, DAHLQUIST, FW, and DROBNY, G. "N-15 AMIDE CHEMICAL-SHIFT TENSORS OF SEVERAL DIPEPTIDES." BIOPHYSICAL JOURNAL 49.2 (February 1986): A328-A328.
Source
wos-lite
Published In
Biophysical Journal
Volume
49
Issue
2
Publish Date
1986
Start Page
A328
End Page
A328

Assignment of proton amide resonances of T4 lysozyme by 13C and 15N multiple isotopic labeling

Authors
Griffey, RH; Redfield, AG; McIntosh, LP; Oas, TG; Dahlquist, FW
MLA Citation
Griffey, RH, Redfield, AG, McIntosh, LP, Oas, TG, and Dahlquist, FW. "Assignment of proton amide resonances of T4 lysozyme by 13C and 15N multiple isotopic labeling." Journal of the American Chemical Society 108.21 (1986): 6816-6817.
Source
scival
Published In
Journal of the American Chemical Society
Volume
108
Issue
21
Publish Date
1986
Start Page
6816
End Page
6817

C-13 CARBONYL CHEMICAL-SHIFT TENSORS OF 3 GLYCINE DIPEPTIDES

Authors
OAS, TG; MCMAHON, TJ; DAHLQUIST, FW; DROBNY, G
MLA Citation
OAS, TG, MCMAHON, TJ, DAHLQUIST, FW, and DROBNY, G. "C-13 CARBONYL CHEMICAL-SHIFT TENSORS OF 3 GLYCINE DIPEPTIDES." BIOPHYSICAL JOURNAL 47.2 (1985): A331-A331.
Source
wos-lite
Published In
Biophysical Journal
Volume
47
Issue
2
Publish Date
1985
Start Page
A331
End Page
A331

COMPARISON OF ASSOCIATIONS OF DIFFERENT HYDROCARBONS WITH CLAY PARTICLES IN SIMULATED SEAWATER.

Some spects of the association of hydrocarbons and smectite clay in simulated seawater were investigated using NaCl solutions in laboratory experiments. Both n-eicosane and n-eicosene displayed identical association behaviors with this clay. Association increased linearly with increasing hydrocarbon concentration in water.

Authors
Meyers, PA; Oas, TG
MLA Citation
Meyers, PA, and Oas, TG. "COMPARISON OF ASSOCIATIONS OF DIFFERENT HYDROCARBONS WITH CLAY PARTICLES IN SIMULATED SEAWATER." Environmental Science and Technology 12.8 (1978): 934-937.
Source
scival
Published In
Environmental Science and Technology
Volume
12
Issue
8
Publish Date
1978
Start Page
934
End Page
937
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Research Areas:

  • Arrestins
  • Binding Sites
  • Binding, Competitive
  • Biophysics
  • Calorimetry
  • Circular Dichroism
  • Computer Simulation
  • Deuterium Exchange Measurement
  • Diffusion
  • Enzyme Stability
  • Hydrogen-Ion Concentration
  • Hydrophobic and Hydrophilic Interactions
  • Kinetics
  • Ligands
  • Magnetic Resonance Spectroscopy
  • Mass Spectrometry
  • Models, Chemical
  • Models, Molecular
  • Models, Statistical
  • Models, Theoretical
  • Molecular Conformation
  • Molecular Structure
  • Motion
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptide Fragments
  • Pliability
  • Protein Binding
  • Protein Conformation
  • Protein Denaturation
  • Protein Folding
  • Protein Stability
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Proteins
  • RNA, Catalytic
  • Receptors, Adrenergic, beta-2
  • Reproducibility of Results
  • Ribonuclease P
  • Ribonucleases
  • Ribonucleoproteins
  • Spectrometry, Fluorescence
  • Spectrum Analysis
  • Staphylococcal Protein A
  • Structural Homology, Protein
  • Structure-Activity Relationship
  • Temperature
  • Thermodynamics
  • Time Factors
  • Titrimetry
  • Viral Proteins