Hydrogen-exchange kinetics in the cold denatured state of ribonuclease A

Nash, David P.; Lee, Bao‐Shiang; Jonáš, J.

doi:10.1016/0167-4838(96)00085-4

Cited by 36 publications

(33 citation statements)

References 20 publications

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“…As discussed in detail in the original studies (17,18) the percent denaturation of each histidine residue can be calculated and a pressure-temperature phase diagram of RNase can be constructed. Figure 3 shows the pressuretemperature phase diagram of RNase A.…”

Section: Resultsmentioning

confidence: 99%

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Cold Denaturation of Proteins

Jonáš

1997

ACS Symposium Series

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By taking advantage of the phase behavior of water, high pressure can significantly lower the freezing point of an aqueous protein solution. In this way, using high-resolution, high-pressure NMR techniques, one can investigate not only pressure denaturation but also cold denaturation of proteins.After an overview of compression effects on dynamic and hydrodynamic behavior of water and heavy water at subzero temperatures, the main part of this contribution is devoted to selected results from recent pressure and cold denaturation NMR studies of Ribonuclease A. The cold denatured state of Ribonuclease A contains partial secondary structures in contrast to its thermally denatured state which contains little or no stable hydrogen bond structures. It was interesting to find that the pattern of protection factors for the pressure and cold denatured states of Ribonuclease A obtained by hydrogen exchange experiments parallels the pattern of protection factors for the folding intermediate of Ribonuclease A reported by Udgoaonkar and Baldwin on the basis of their pulsed hydrogen experiments.Increasing attention has recently been focused on denatured and partially folded states, since determination of their structure and stability .may provide novel information for the mechanisms of protein folding (1-3). The native conformations of hundreds of proteins are known in great detail from structural determinations by X-ray crystallography and, more recently, NMR spectroscopy. However, a detailed knowledge of the conformations of denatured and partially folded states is lacking, and represents a serious shortcoming in current studies of protein stability and protein folding pathways (2).Protein folding, the relationship between the amino acid sequence and the 310

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Section: Resultsmentioning

confidence: 99%

“…Several recent studies performed in our laboratory (16)(17)(18)(19)(20)(21) illustrate the unique information obtained by combining high-resolution NMR techniques with high pressure.…”

Section: Cold Denaturation Ofproteins 311mentioning

confidence: 96%

Cold Denaturation of Proteins

Jonáš

1997

ACS Symposium Series

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“…This was confirmed by NMR experiments on Ribonuclease A, where the protection factors for the H/D exchange were similar for the cold and the pressure unfolded states. These states were also found to be more compact than the heat unfolded structure (Nash et al 1996).…”

Section: Thermodynamic Description Of the Two State Foldingmentioning

confidence: 94%

“…Since that, many pressure experiments were performed on different types of ribonucleases (Yamaguchi et al 1995;Nash et al 1996;Yamasaki et al 1998;Ribo et al 2006;Zhai and Winter 2013). Jonas's group compared the cold, heat and pressure denaturation of ribonuclease using NMR spectroscopy (Zhang et al 1995).…”

Section: Ribonucleasementioning

confidence: 99%

Protein Denaturation on p-T Axes – Thermodynamics and Analysis

Smeller

2015

Subcellular Biochemistry

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Proteins are essential players in the vast majority of molecular level life processes. Since their structure is in most cases substantial for their correct function, study of their structural changes attracted great interest in the past decades. The three dimensional structure of proteins is influenced by several factors including temperature, pH, presence of chaotropic and cosmotropic agents, or presence of denaturants. Although pressure is an equally important thermodynamic parameter as temperature, pressure studies are considerably less frequent in the literature, probably due to the technical difficulties associated to the pressure studies. Although the first steps in the high-pressure protein study have been done 100 years ago with Bridgman's ground breaking work, the field was silent until the modern spectroscopic techniques allowed the characterization of the protein structural changes, while the protein was under pressure. Recently a number of proteins were studied under pressure, and complete pressure-temperature phase diagrams were determined for several of them. This review summarizes the thermodynamic background of the typical elliptic p-T phase diagram, its limitations and the possible reasons for deviations of the experimental diagrams from the theoretical one. Finally we show some examples of experimentally determined pressure-temperature phase diagrams.

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“…Cold denatured apomyoglobin at Ϫ5°C exhibits high ellipticity, Ϸ70% of the native value at 222 nm (61). The persistence of helical structure in cold denatured proteins appears to be widespread (63)(64)(65). Molecules that start with preformed structure, past the initial nucleation step, can be expected to fold much faster than the millisecond nucleation rate (2).…”

Section: )mentioning

confidence: 99%

Ultrafast signals in protein folding and the polypeptide contracted state

Sosnick

Shtilerman

Mayne

et al. 1997

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

165

155

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To test the significance of ultrafast protein folding signals (< <1 msec), we studied cytochrome c (Cyt c) and two Cyt c fragments with major C-terminal segments deleted. The fragments remain unfolded under all conditions and so could be used to define the unfolded baselines for protein f luorescence and circular dichroism ( Many recent publications describe submillisecond foldingrelated signals (1) and interpret these signals in terms of the rapid formation of productive folding intermediates. Our earlier results lead us to question this interpretation. We found that the folding rate of cytochrome c (Cyt c) is limited by an initial, time-requiring, nucleated polypeptide chain collapse (2). The nucleated collapse represents the rate-limiting step for overall folding when folding is two-state and it limits the rate at which intermediates can populate when folding is more than two-state, i.e., when an even larger misfold-reorganization barrier is encountered in subsequent folding (3). The nucleated collapse itself appears to be limited by a large scale diffusion-dependent conformational search, culminating in the assembly of a transition state nucleus that can support forward folding steps in an energetically downhill manner. For Cyt c the initial search-nucleation step requires about 1 msec under the most favorable conditions, and results available for other small proteins when starting from the fully unfolded state seemed to us to be generally consistent with a similar requirement (2). However, this view of the strategy for the initiation of folding now appears to be contradicted by many recent reports of much faster folding events, both in Cyt c and other proteins.This paper considers the significance of ultrafast protein folding signals. The results show that when unfolded Cyt c is diluted from high denaturant in folding experiments, it experiences a fast polymer chain contraction from a more extended form of the unfolded state to a more contracted form of the unfolded state. The optical signals produced by the chain contraction can misleadingly mimic the formation of a distinct folding intermediate. Rather, the supposed intermediate is the unfolded state itself.An assessment of available fast folding observations in this light shows them to be consistent with the view that folding is limited by an obligatory, initial, time-requiring (Ϸ1 msec) search-nucleation step. MATERIALS AND METHODSHorse heart Cyt c (highest available grade) was from Sigma Chemical Company. Cyt c fragments were produced by partially selective cyanogen bromide cleavage (4) at the methionine residues, Met-65 and -80, purified by reverse phase HPLC (C18 column, acetonitrile gradient), and verified by mass spectrometry. F1-65 was virtually pure. The F1-80 preparation was contaminated (Ϸ10%) with a species close to Cyt c in mass. Molar concentration of the fragments was measured by absorbance using an extinction coefficient at 401 nm of 137,000 M Ϫ1 ⅐cm Ϫ1 in denaturing guanidinium chloride (GdmCl), obtained by comparison with holo Cy...

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Hydrogen-exchange kinetics in the cold denatured state of ribonuclease A

Cited by 36 publications

References 20 publications

Cold Denaturation of Proteins

Cold Denaturation of Proteins

Protein Denaturation on p-T Axes – Thermodynamics and Analysis

Ultrafast signals in protein folding and the polypeptide contracted state

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