Rapid Sample-Mixing Technique for Transient NMR and Photo-CIDNP Spectroscopy:  Applications to Real-Time Protein Folding

Mok, K. Hun; Nagashima, Toshio; Day, Iain J.; Jones, Jonathan A.; Jones, Charles J. V.; Dobson, Christopher M.; Hore, P. J.

doi:10.1021/ja036357v

Cited by 100 publications

(107 citation statements)

References 48 publications

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“…The triggering of kinetic reactions can be achieved either by a sudden change of the protein state itself, e.g., by a photo-induced excitation or cleavage of chemical bonds (16,17), or by a change of environment, such as the solvent composition, temperature, or pH (4,18). Here, we opted for an initiation of the reaction by an abrupt change in the solvent conditions achieved by rapid mixing of two solutions inside the NMR magnet (5,19). The fast mixing device used for this work (SI Fig.…”

Section: Resultsmentioning

confidence: 99%

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.

152

151

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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: Resultsmentioning

confidence: 99%

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.

152

151

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“…We have recently developed NMR techniques to characterize aromatic side chains in transient, kinetic intermediate species present in real-time protein folding experiments (13,14) or in partially folded states that are stable under equilibrium conditions (15,16). To circumvent the difficulties in studying such states directly, we have shown that magnetization transfer from the partially folded state to the N state by rapidly refolding the protein can, in principle, yield structural information on the former from the well resolved spectrum of the latter (14)(15)(16).…”

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confidence: 99%

“…To circumvent the difficulties in studying such states directly, we have shown that magnetization transfer from the partially folded state to the N state by rapidly refolding the protein can, in principle, yield structural information on the former from the well resolved spectrum of the latter (14)(15)(16). However, to carry out such experiments successfully, it is essential that refolding takes place faster than nuclear spin-lattice relaxation.…”

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confidence: 99%

Multiple subsets of side-chain packing in partially folded states of α-lactalbumins

Mok

Nagashima

Day

et al. 2005

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

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Photochemically induced dynamic nuclear polarization NMR pulselabeling techniques have been used to obtain detailed information about side-chain surface accessibilities in the partially folded (molten globule) states of bovine and human ␣-lactalbumin prepared under a variety of well defined conditions. Pulse labeling involves generating nuclear polarization in the partially folded state, rapidly refolding the protein within the NMR sample tube, then detecting the polarization in the well dispersed native-state spectrum. Differences in the solvent accessibility of specific side chains in the various molten globule states indicate that the hydrophobic clusters involved in stabilizing the ␣-lactalbumin fold can be formed from interactions between a variety of different hydrophobic residues in both native and nonnative environments. The multiple subsets of hydrophobic clusters are likely to result from the existence of distinct but closely related local minima on the free-energy landscape of the protein and show that the fold and topology of a given protein may be formed from degenerate groups of side chains.hydrophobic cluster ͉ protein folding ͉ pulse labeling ͉ chemically induced dynamic nuclear polarization ͉ molten globule V ery significant advances, both theoretical and experimental, have been made in recent years in elucidating the principles that govern the folding of proteins (1, 2). In particular, the concept of an energy landscape has provided a general framework for interpreting the thermodynamics and kinetics of the folding process through which a polypeptide chain converts from a disorganized unfolded state into the tightly packed native state (N state) at the global energy minimum (3, 4). Recent approaches, involving a combination of computer-simulation techniques with experimental constraints, have enabled three-dimensional structural ensembles of various partially folded species and folding intermediates to be defined and provide a coarse-grained description of the development of interresidue interactions and chain topologies involved in attaining the final native structure (5-7). Of particular importance in this context are compact denatured states, often known as molten globules (MGs), which are commonly characterized by the presence of native-like secondary structure and hydrodynamic radii but lack both native-like packing of the internal amino acid side chains and exclusion of solvent molecules from the hydrophobic core of the protein (8, 9). The detailed experimental description of such states will undoubtedly provide important information about the specific factors stabilizing the overall chain topology at a residue-specific side-chain level (10). However, MG states are very difficult to characterize by using conventional x-ray crystallography or NMR techniques because of their heterogeneous character (11,12).We have recently developed NMR techniques to characterize aromatic side chains in transient, kinetic intermediate species present in real-time protein folding experiments (13, 14) or in p...

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“…[33][34][35] The combination of atomic resolution applicable to relatively fast kinetics cannot be achieved by any other biophysical method. Dynamic events of biomacromolecules in this time range are large scale conformational transitions, folding events (for proteins) and macromolecular assembly.…”

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confidence: 99%

“…82 For the latter experimental setup, there are also two possibilities amenable: in the first method one solution is injected into another inside the NMR tube, so that after the injection the turbulences cause homogeneous and rapid mixing. 33 The second method uses mobile separators of the two solutions that can be removed rapidly and thereby facilitate mixing. 83 For the very early mixing instruments, already after a dead time of s dead $ 0.2 s a spectrum with sufficient resolution could be recorded.…”

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confidence: 99%

Time‐resolved NMR studies of RNA folding

et al. 2007

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The application of real‐time NMR experiments to the study of RNA folding, as reviewed in this article, is relatively new. For many RNA folding events, current investigations suggest that the time scales are in the second to minute regime. In addition, the initial investigations suggest that different folding rates are observed for one structural transition may be due to the hierarchical folding units of RNA. Many of the experiments developed in the field of NMR of protein folding cannot directly be transferred to RNA: hydrogen exchange experiments outside the spectrometer cannot be applied since the intrinsic exchange rates are too fast in RNA, relaxation dispersion experiments on the other require faster structural transitions than those observed in RNA. On the other hand, information derived from time‐resolved NMR experiments, namely the acquisition of native chemical shifts, can be readily interpreted in light of formation of a single long‐range hydrogen bonding interaction. Together with mutational data that can readily be obtained for RNA and new ligation technologies that enhance site resolution even further, time‐resolved NMR may become a powerful tool to decipher RNA folding. Such understanding will be of importance to understand the functions of coding and non‐coding RNAs in cells. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 360–383, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Rapid Sample-Mixing Technique for Transient NMR and Photo-CIDNP Spectroscopy: Applications to Real-Time Protein Folding

Cited by 100 publications

References 48 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

Multiple subsets of side-chain packing in partially folded states of α-lactalbumins

Time‐resolved NMR studies of RNA folding

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