A hypothesis to reconcile the physical and chemical unfolding of proteins (original) (raw)
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Angewandte Chemie, 2015
The energetic and volumetric properties of a threestate protein folding system, comprising a metastable triple mutant of the Fyn SH3 domain, have been investigated using pressure-dependent 15 N-relaxation dispersion NMR from 1 to 2500 bar. Changes in partial molar volumes (DV) and isothermal compressibilities (Dk T ) between all the states along the folding pathway have been determined to reasonable accuracy. The partial volume and isothermal compressibility of the folded state are 100 mL mol À1 and 40 mL mol À1 bar À1 , respectively, higher than those of the unfolded ensemble. Of particular interest are the findings related to the energetic and volumetric properties of the on-pathway folding intermediate. While the latter is energetically close to the unfolded state, its volumetric properties are similar to those of the folded protein.
The use of high-pressure nuclear magnetic resonance to study protein folding
Methods in molecular biology (Clifton, N.J.), 2007
Recent development of high-pressure cells for a variety of spectroscopic methods has enabled the use of pressure as one of the commonly used perturbations along with temperature and chemical perturbations to study folding/unfolding reactions of proteins. Although various high-pressure spectroscopy techniques have their own significance, high-pressure nuclear magnetic resonance (NMR) is unique in that it allows one to gain residue-specific and atom-detailed information from proteins under pressure. Furthermore, because of a peculiar volume property of a protein, high-pressure NMR allows one to obtain structural information of a protein in a wide conformational space from the bottom to the upper region of the folding funnel, giving structural reality for the "open" state of a protein proposed from hydrogen exchange. The method allows a link between equilibrium folding intermediates and the kinetic intermediates, and manifests a new view of proteins as dynamic entities amply ...
High-pressure NMR techniques for the study of protein dynamics, folding and aggregation
Journal of magnetic resonance (San Diego, Calif. : 1997), 2017
High-pressure is a well-known perturbation method used to destabilize globular proteins and dissociate protein complexes or aggregates. The heterogeneity of the response to pressure offers a unique opportunity to dissect the thermodynamic contributions to protein stability. In addition, pressure perturbation is generally reversible, which is essential for a proper thermodynamic characterization of a protein equilibrium. When combined with NMR spectroscopy, hydrostatic pressure offers the possibility of monitoring at an atomic resolution the structural transitions occurring upon unfolding and determining the kinetic properties of the process. The recent development of commercially available high-pressure sample cells greatly increased the potential applications for high-pressure NMR experiments that can now be routinely performed. This review summarizes the recent applications and future directions of high-pressure NMR techniques for the characterization of protein conformational flu...
Cavities determine the pressure unfolding of proteins
Proceedings of the National Academy of Sciences, 2012
It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavitycontaining variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches. energy landscape | fluorescence | volume change T he first observation that pressure unfolds proteins was made in 1914 by Bridgman (1). Despite numerous studies since then, the physical basis of the pressure-induced unfolding of proteins has not been explained. This difference in volume underlying pressure effects has been rationalized previously in terms of (i) increases in solvent density concomitant with solvation of exposed surfaces upon unfolding (2), (ii) modifications in the structure of bulk water leading to weakened hydrophobic interactions (3), and (iii) cavities in the folded state that are not present in the unfolded state (4-7). The goal of this study was to examine the structural origins of pressure unfolding of proteins. Twenty-five years ago Walter Kauzmann stressed the importance of understanding pressure effects : "Enthalpy and volume are equally fundamental properties of the (protein) unfolding process, and no model can be considered acceptable unless it accounts for the entire thermodynamic behavior." He also noted important discrepancies between the volumetric properties of hydrophobic interactions and the pressure unfolding of proteins.
Pressure-induced protein-folding/unfolding kinetics
Proceedings of the …, 1999
We use an off-lattice minimalist model to describe the effects of pressure in slowing down the folding͞unfolding kinetics of proteins when subjected to increasingly larger pressures. The potential energy function used to describe the interactions between beads in the model includes the effects of pressure on the pairwise interaction of hydrophobic groups in water. We show that pressure affects the participation of contacts in the transition state. More significantly, pressure exponentially decreases the chain reconfigurational diffusion coefficient. These results are consistent with experimental results on the kinetics of pressure-denaturation of staphylococcal nuclease.
Dynamics and folding of proteins are intimately related to their volumetric properties, such as volume and compressibility associated with conformational states. These properties can only be explored in experiments in which pressure is a variable, Here, site-specific analysis of compressibility and stability of a protein is a major current concern from the viewpoint of structural biology. Combination of the on-line high pressure cell technique with a commercial high field NMR spectrometer (e.g., 750 MHz for proton) paves the way to the solution. Pressure-sensitive, flexible or fragile regions of a protein can be identified in this manner by IH, 15N and 13C NMR spectroscopy under varying hydrostatic pressure between I and similar to4 kbar. Pressure-induced changes of chemical shifts of essentially all the 1H and 15N NMR signals along with changes in IH nuclear Overhauser effect (NOE) reveal that the entire folded structure responds sensitively to pressure. However, the response can b...
2020
ABSTRACTProteins often interconvert between different conformations in ways critical to their function. While manipulating such equilibria for biophysical study is often challenging, the application of pressure is a potential route to achieve such control by favoring the population of lower volume states. Here, we use this feature to study the interconversion of ARNT PAS-B Y456T, which undergoes a dramatic beta-strand slip as it switches between two stably-folded conformations. Coupling high pressure and biomolecular NMR, we obtained the first quantitative data testing two key hypotheses of this process: the slipped conformation is both smaller and less compressible than the wildtype equivalent, and the interconversion proceeds through a chiefly-unfolded intermediate state. Our work exemplifies how these approaches, which can be generally applied to protein conformational switches, can provide unique information that is not easily accessible through other techniques.
Exploring Early Stages of the Chemical Unfolding of Proteins at the Proteome Scale
PLoS Computational Biology, 2013
After decades of using urea as denaturant, the kinetic role of this molecule in the unfolding process is still undefined: does urea actively induce protein unfolding or passively stabilize the unfolded state? By analyzing a set of 30 proteins (representative of all native folds) through extensive molecular dynamics simulations in denaturant (using a range of forcefields), we derived robust rules for urea unfolding that are valid at the proteome level. Irrespective of the protein fold, presence or absence of disulphide bridges, and secondary structure composition, urea concentrates in the first solvation shell of quasi-native proteins, but with a density lower than that of the fully unfolded state. The presence of urea does not alter the spontaneous vibration pattern of proteins. In fact, it reduces the magnitude of such vibrations, leading to a counterintuitive slow down of the atomic-motions that opposes unfolding. Urea stickiness and slow diffusion is, however, crucial for unfolding. Long residence urea molecules placed around the hydrophobic core are crucial to stabilize partially open structures generated by thermal fluctuations. Our simulations indicate that although urea does not favor the formation of partially open microstates, it is not a mere spectator of unfolding that simply displaces to the right of the foldedrRunfolded equilibrium. On the contrary, urea actively favors unfolding: it selects and stabilizes partially unfolded microstates, slowly driving the protein conformational ensemble far from the native one and also from the conformations sampled during thermal unfolding.
Unique Features of the Folding Landscape of a Repeat Protein Revealed by Pressure Perturbation
Biophysical Journal, 2010
The volumetric properties of proteins yield information about the changes in packing and hydration between various states along the folding reaction coordinate and are also intimately linked to the energetics and dynamics of these conformations. These volumetric characteristics can be accessed via pressure perturbation methods. In this work, we report high-pressure unfolding studies of the ankyrin domain of the Notch receptor (Nank1-7) using fluorescence, small-angle x-ray scattering, and Fourier transform infrared spectroscopy. Both equilibrium and pressure-jump kinetic fluorescence experiments were consistent with a simple two-state folding/unfolding transition under pressure, with a rather small volume change for unfolding compared to proteins of similar molecular weight. High-pressure fluorescence, Fourier transform infrared spectroscopy, and small-angle x-ray scattering measurements revealed that increasing urea over a very small range leads to a more expanded pressure unfolded state with a significant decrease in helical content. These observations underscore the conformational diversity of the unfolded-state basin. The temperature dependence of pressure-jump fluorescence relaxation measurements demonstrated that at low temperatures, the folding transition state ensemble (TSE) lies close in volume to the folded state, consistent with significant dehydration at the barrier. In contrast, the thermal expansivity of the TSE was found to be equivalent to that of the unfolded state, indicating that the interactions that constrain the folded-state thermal expansivity have not been established at the folding barrier. This behavior reveals a high degree of plasticity of the TSE of Nank1-7. FIGURE 3 (A and B) Pressure-dependent pair distribution functions obtained from analysis of the SAXS data on Nank1-7 at 24 C at 2 M (A) and 2.2 M (B) urea. Pressures are as indicated in the figures. (C) Comparison of the pair-distribution functions at 1 bar and 0 M urea (red line), and at 3 kbar in the presence of 0 M urea (yellow dotted line), 2 M urea (green dotted line), and 2.2 M urea (blue line).