June 2012
Publication year: 2012 Source:Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Volume 1824, Issue 6
The transient unfolding events from the native state of a protein towards higher energy states can be closely investigated by studying the process of hydrogen exchange. Here, we present BLUU-Tramp (Biophysics Laboratory University of Udine—Temperature ramp), a new method to measure the rates for the exchange process and the underlying equilibrium thermodynamic parameters, using just a single sample preparation, in a single experiment that lasts some 20 to 60h depending on the protein thermal stability, to record hundreds of points over a virtually continuous temperature window. The method is suitable also in presence of other proteins in the sample, if only the target protein is 15N-labelled. This allows the complete thermodynamic description of the unfolding landscape at an atomic level in the presence of small or macromolecular ligands or cosolutes, or in physiological environments. The method was successfully tested with human ubiquitin. Then the unfolding thermodynamic parameters were satisfactorily determined for the amyloidogenic protein ?2-microglobulin, in aqueous buffer and in synovial liquid, that is the natural medium of amyloid deposition in joints. Graphical abstract
Highlights
? NMR hydrogen–deuterium exchange measurements to study protein unfolding landscape. ? BLUU-Tramp, new NMR method to measure isotope exchange thermal kinetics. ? BLUU-Tramp: time gain, single sample and experimental session, routine instruments. ? Identification of stability determinants in proteins and protein complexes. ? Application to amyloidogenic systems, physiological milieu, basic folding studies.
Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques: TFE-induced unfolding of KcsA in DDM surfactant
Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques: TFE-induced unfolding of KcsA in DDM surfactant
September 2012
Publication year: 2012
Source:Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1818, Issue 9</br>
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Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes...
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Long-Lived States to Monitor Protein Unfolding by Proton NMR.
Long-Lived States to Monitor Protein Unfolding by Proton NMR.
Long-Lived States to Monitor Protein Unfolding by Proton NMR.
Chemphyschem. 2011 Aug 31;
Authors: Bornet A, Ahuja P, Sarkar R, Fernandes L, Hadji S, Lee SY, Haririnia A, Fushman D, Bodenhausen G, Vasos PR
Abstract
The relaxation of long-lived states (LLS) corresponds to the slow return to statistical thermal equilibrium between symmetric and antisymmetric proton spin states. This process is remarkably sensitive to the presence of external spins and can be used to obtain...
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09-02-2011 05:40 PM
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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05-31-2011 11:41 PM
Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion.
Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion.
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)...
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05-13-2011 02:40 PM
[NMR paper] Insights into conformation and dynamics of protein GB1 during folding and unfolding b
Insights into conformation and dynamics of protein GB1 during folding and unfolding by NMR.
Related Articles Insights into conformation and dynamics of protein GB1 during folding and unfolding by NMR.
J Mol Biol. 2004 Jan 30;335(5):1299-307
Authors: Ding K, Louis JM, Gronenborn AM
Understanding protein stability requires characterization of structural determinants of the folded and unfolded states. Many proteins are capable of populating partially folded states under specific solution conditions. Occasionally, coexistence of the folded and an...
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11-24-2010 09:25 PM
[NMR paper] Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate reveal
Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering.
Related Articles Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering.
J Mol Biol. 2002 Sep 27;322(4):841-9
Authors: Fowler SB, Best RB, Toca Herrera JL, Rutherford TJ, Steward A, Paci E, Karplus M, Clarke J
The mechanical unfolding of an immunoglobulin domain from the...
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11-24-2010 08:58 PM
[NMR paper] Measurement of long-range cross-correlation rates using a combination of single- and
Measurement of long-range cross-correlation rates using a combination of single- and multiple-quantum NMR spectroscopy in one experiment.
Related Articles Measurement of long-range cross-correlation rates using a combination of single- and multiple-quantum NMR spectroscopy in one experiment.
J Am Chem Soc. 2002 Apr 17;124(15):4050-7
Authors: Fruh D, Chiarparin E, Pelupessy P, Bodenhausen G
A method is described to determine long-range cross-correlations between the modulations of an anisotropic chemical shift (e.g., of a C' carbonyl carbon in...
Single-shot NMR measurement of protein unfolding landscapes
Highlights
Linear Mode
