Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion.
Proc Natl Acad Sci U S A. 2011 May 11;
Authors: Meinhold DW, Wright PE
Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use (15)N, , and (13)CO NMR R(2) relaxation dispersion to investigate spontaneous unfolding and refolding events of native apomyoglobin. Above pH 5.0, dispersion is dominated by processes involving fluctuations of the F-helix region, which is invisible in NMR spectra. Measurements of R(2) dispersion for residues contacted by the F-helix region in the native (N) structure reveal a transient state formed by local unfolding of helix F and undocking from the protein core. A similar state was detected at pH 4.75-4.95 and determined to be an on-pathway intermediate (I1) in a linear three-state unfolding scheme (N&lrarr2;I1&lrarr2;MG) leading to a transiently populated molten globule (MG) state. The slowest steps in unfolding and refolding are N*->*I1 (36*s(-1)) and MG*->*I1 (26*s(-1)), respectively. Differences in chemical shift between N and I1 are very small, except in regions adjacent to helix F, showing that their core structures are similar. Chemical shift changes between the N and MG states, obtained from R(2) dispersion, reveal that the transient MG state is structurally similar to the equilibrium MG observed previously at high temperature and low pH. Analysis of MG state chemical shifts shows the location of residual helical structure in the transient intermediate and identifies regions that unfold or rearrange into nonnative structure during the N*->*MG transition. The experiments also identify regions of energetic frustration that "crack" during unfolding and impede the refolding process.
PMID: 21562212 [PubMed - as supplied by publisher]
Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion [Biophysics and Computational Biology]
Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion
Meinhold, D. W., Wright, P. E....
Date: 2011-05-31
Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use 15N, , and 13CO NMR R2 relaxation dispersion to investigate spontaneous unfolding and refolding events of native apomyoglobin. Above pH 5.0, dispersion is dominated by processes involving fluctuations of the F-helix region, which...
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Structure, Dynamics, and Kinetics of Weak Protein-Protein Complexes from NMR Spin Relaxation Measurements of Titrated Solutions.
Structure, Dynamics, and Kinetics of Weak Protein-Protein Complexes from NMR Spin Relaxation Measurements of Titrated Solutions.
Structure, Dynamics, and Kinetics of Weak Protein-Protein Complexes from NMR Spin Relaxation Measurements of Titrated Solutions.
Angew Chem Int Ed Engl. 2011 Mar 18;
Authors: Salmon L, Ortega Roldan JL, Lescop E, Licinio A, van Nuland N, Jensen MR, Blackledge M
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03-23-2011 05:41 PM
Measuring (1)H (N) temperature coefficients in invisible protein states by relaxation dispersion NMR spectroscopy.
Measuring (1)H (N) temperature coefficients in invisible protein states by relaxation dispersion NMR spectroscopy.
Measuring (1)H (N) temperature coefficients in invisible protein states by relaxation dispersion NMR spectroscopy.
J Biomol NMR. 2011 Mar 18;
Authors: Bouvignies G, Vallurupalli P, Cordes MH, Hansen DF, Kay LE
A method based on the Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for measuring the temperature coefficients of amide proton chemical shifts of low populated 'invisible' protein states that exchange...
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03-23-2011 05:41 PM
[NMR paper] NMR spectroscopic characterization of millisecond protein folding by transverse relaxation dispersion measurements.
NMR spectroscopic characterization of millisecond protein folding by transverse relaxation dispersion measurements.
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The cold shock protein CspB adopts its native and functional tertiary structure on the millisecond time scale. We employed transverse relaxation NMR methods, which allow a quantitative measurement of the cooperativity of this...
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Nature. 2004 Jul 29;430(6999):586-90
Authors: Korzhnev DM, Salvatella X, Vendruscolo M, Di Nardo AA, Davidson AR, Dobson CM, Kay LE
Many biochemical processes proceed through the formation of functionally significant intermediates. Although the identification and characterization of such species can provide vital clues about the mechanisms of the...
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[NMR paper] Unfolding kinetics of tryptophan side chains in the dimerization and hinge regions of
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HIV I protease has been the target of extensive and variety of investigations in recent years because of its importance in the AIDS viral life cycle. We...
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[NMR paper] NMR analysis of staphylococcal nuclease thermal quench refolding kinetics.
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http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www3.interscience.wiley.com-aboutus-images-wiley_interscience_pubmed_logo_FREE_120x27.gif http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www.pubmedcentral.nih.gov-corehtml-pmc-pmcgifs-pubmed-pmc.gif Related Articles NMR analysis of staphylococcal nuclease thermal quench refolding kinetics.
Protein Sci. 1993 May;2(5):851-8
Authors: Kautz RA, Fox RO
Thermally unfolded staphylococcal nuclease has been rapidly...
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08-21-2010 11:53 PM
Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively 13C labeled samples
Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively 13C labeled samples
Patrik Lundström, D. Flemming Hansen and Lewis E. Kay
Journal of Biomolecular NMR; 2008; 42(1); pp 35 - 47
Abstract: Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for quantifying chemical shifts of excited protein states. For many applications of the technique that involve the measurement of relaxation rates of carbon...
Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion.
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