Following protein folding in real time using NMR spectroscopy

Balbach, Jochen; Forge, Vincent; Nuland, Nico A. J. van; Winder, Steve L.; Hore, P. J.; Dobson, Christopher M.

doi:10.1038/nsb1095-865

Cited by 238 publications

(208 citation statements)

References 30 publications

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“…One-dimensional 1 H NMR spectra recorded during the refolding reaction of apo-␣-lactalbumin provided evidence that the MG state, accumulated during the early stages of the folding reaction, shows similar spectral characteristics (poorly dispersed and broad resonances) and therefore similar dynamic properties of the conformational ensemble to those observed for the MG state stabilized at acidic pH (25). Additional diffusion-edited and NOE transfer NMR measurements allowed monitoring the compactness of the protein and the establishing of native tertiary contacts, respectively (23,27).…”

Section: Conformational Transition Kinetics Of ␣-Lactalbumin From An mentioning

confidence: 83%

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

Schanda

Forge

Brutscher

2007

Proc. Natl. Acad. Sci. U.S.A.

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152

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Atom-resolved real-time studies of kinetic processes in proteins have been hampered in the past by the lack of experimental techniques that yield sufficient temporal and atomic resolution. Here we present band-selective optimized flip-angle short transient (SOFAST) real-time 2D NMR spectroscopy, a method that allows simultaneous observation of reaction kinetics for a large number of nuclear sites along the polypeptide chain of a protein with an unprecedented time resolution of a few seconds. SOFAST real-time 2D NMR spectroscopy combines fast NMR data acquisition techniques with rapid sample mixing inside the NMR magnet to initiate the kinetic event. We demonstrate the use of SOFAST real-time 2D NMR to monitor the conformational transition of ␣-lactalbumin from a molten globular to the native state for a large number of amide sites along the polypeptide chain. The kinetic behavior observed for the disappearance of the molten globule and the appearance of the native state is monoexponential and uniform along the polypeptide chain. This observation confirms previous findings that a single transition state ensemble controls folding of ␣-lactalbumin from the molten globule to the native state. In a second application, the spontaneous unfolding of native ubiquitin under nondenaturing conditions is characterized by amide hydrogen exchange rate constants measured at high pH by using SOFAST real-time 2D NMR. Our data reveal that ubiquitin unfolds in a gradual manner with distinct unfolding regimes.hydrogen/deuterium exchange ͉ molecular kinetics ͉ ␣-lactalbumin ͉ ubiquitin A detailed description of the structural changes occurring during the folding and unfolding of proteins still remains a challenging objective in biophysics. The understanding of the fundamental mechanisms of the folding process will also shed light on the factors leading to protein misfolding responsible for neurodegenerative diseases such as Alzheimer's and Parkinson's diseases (1). The ideal method to study protein folding/unfolding provides structural information at atomic resolution and is sensitive to changes occurring on time scales ranging from microseconds to minutes, a task that no single technique is able to fulfill. NMR spectroscopy is especially well adapted to obtain detailed atomistic information about the mechanisms, kinetics, and energetics of the folding/unfolding process for virtually every nuclear site in the protein. NMR methods are sensitive to molecular dynamics occurring over a wide range of time scales (Fig. 1a). Although steady-state NMR methods are well suited to characterize equilibrium molecular dynamics occurring on a subsecond time scale (2, 3), unidirectional processes are best studied by real-time NMR (4, 5), where a series of NMR spectra is recorded during the reaction (Fig. 1b). Two difficulties have so far hampered the widespread application of real-time NMR to the study of protein folding. The first problem is the low intrinsic sensitivity of NMR at ambient temperature, a consequence of the small transition energies ...

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Section: Conformational Transition Kinetics Of ␣-Lactalbumin From An mentioning

confidence: 83%

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

Schanda

Forge

Brutscher

2007

Proc. Natl. Acad. Sci. U.S.A.

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152

151

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“…Two alternative models of the BLA folding It has been known that the molten globule intermediate accumulates at an early stage of the folding of both BLA and 2CM-3SS-BLA and that the subsequent folding process is explained by the two-state process without any other stable intermediates, except for a minor slow folding phase observed in the presence of Ca2+ (Kuwajima et al, 1985Ikeguchi et al, 1986Ikeguchi et al, , 1992Balbach et al, 1995Balbach et al, , 1996Arai & Kuwajima, 1996). In this study, we ignore this minor slow phase, because its amplitude is only 10% of the total observable change during the folding and it is ascribable to a slow isomerization process in the unfolded state .…”

Section: Resultsmentioning

confidence: 99%

“…To explain the bendings in the plot of log k , and log k-vs. GdnHCl concentration, intermediates other than the A state must be incorporated into model 1. However, there is no evidence for the presence of such intermediates, according to the kinetic folding studies of apo BLA investigated by various techniques including UV absorption, fluorescence, CD, pulse hydrogen exchange (Arai & Kuwajima, 1996), real-time observation of appearance of the NMR signal specific to the N state (Balbach et al, 1995), and 2D NMR observation of folding at individual residue levels (Balbach et al, 1996). All of these experiments show that the observable folding kinetics is represented by a single exponential process with the same rate constant independent of the probe used.…”

Section: Is the Molten Globule State Obligatory For Bla Folding?mentioning

confidence: 99%

Transition state in the folding of α‐lactalbumin probed by the 6‐120 disulfide bond

et al. 1998

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The guanidine hydrochloride concentration dependence of the folding and unfolding rate constants of a derivative of a-lactalbumin, in which the 6-120 disulfide bond is selectively reduced and S-carboxymethylated, was measured and compared with that of disulfide-intact a-lactalbumin. The concentration dependence of the folding and unfolding rate constants was analyzed on the basis of the two alternative models, the intermediate-controlled folding model and the multiple-pathway folding model, that we had proposed previously. All of the data supported the multiple-pathway folding model. Therefore, the molten globule state that accumulates at an early stage of folding of a-lactalbumin is not an obligatory intermediate. The cleavage of the 6-120 disulfide bond resulted in acceleration of unfolding without changing the refolding rate, indicating that the loop closed by the 6-120 disulfide bond is unfolded in the transition state. It is theoretically shown that the chain entropy gain on removing the cross-link from a random coil chain with helical stretches can be comparable to that from an entirely random chain. Therefore, the present result is not inconsistent with the known structure in the molten globule intermediate. Based on this result and other knowledge obtained so far, the structure in the transition state of the folding reaction of a-lactalbumin is discussed.

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“…The perturbation may be the addition of a binding partner or denaturant, or a change of temperature. Real-time NMR has been used in a number of seminal studies on cytochrome C (Roder et al 1988), ribonuclease A (Udgaonkar & Baldwin, 1990), and also other proteins (Balbach et al 1995;Harper et al 2004;Haupt et al 2011;Hoeltzli & Frieden, 1995;Udgaonkar & Baldwin, 1990) and RNA (Cao et al 2010;Wenter et al 2005). A direct approach based on using NMR to assess slow dynamics of binding and folding was reported by Binolfi et al (2012), that followed the binding and folding of a truncated version of thioredoxin (residues 1-93, TRX1-93) upon addition of a peptide containing the C-terminal region of the full protein (residues 94-108, TRX94-108).…”

Section: H/d-exchangementioning

confidence: 99%

Protein dynamics and function from solution state NMR spectroscopy

Kovermann

Rogne

Wolf‐Watz

2016

Quart. Rev. Biophys.

148

122

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Abstract. It is well-established that dynamics are central to protein function; their importance is implicitly acknowledged in the principles of the Monod, Wyman and Changeux model of binding cooperativity, which was originally proposed in 1965. Nowadays the concept of protein dynamics is formulated in terms of the energy landscape theory, which can be used to understand protein folding and conformational changes in proteins. Because protein dynamics are so important, a key to understanding protein function at the molecular level is to design experiments that allow their quantitative analysis. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited for this purpose because major advances in theory, hardware, and experimental methods have made it possible to characterize protein dynamics at an unprecedented level of detail. Unique features of NMR include the ability to quantify dynamics (i) under equilibrium conditions without external perturbations, (ii) using many probes simultaneously, and (iii) over large time intervals. Here we review NMR techniques for quantifying protein dynamics on fast (ps-ns), slow (μs-ms), and very slow (s-min) time scales. These techniques are discussed with reference to some major discoveries in protein science that have been made possible by NMR spectroscopy.

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Following protein folding in real time using NMR spectroscopy

Cited by 238 publications

References 30 publications

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

Transition state in the folding of α‐lactalbumin probed by the 6‐120 disulfide bond

Protein dynamics and function from solution state NMR spectroscopy

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