Pressure perturbation calorimetric studies of the solvation properties and the thermal unfolding of proteins in solution—experiments and theoretical interpretation

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483

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 (8): "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.To date, no conclusive explanation for the origins of pressure unfolding of proteins has been proposed. In the present work, 10 cavity-containing variants of staphylococcal nuclease (SNase) were engineered by substitution of internal core residues to Ala. Crystal structures of the variants were obtained to verify the exi...

Section: Discussionmentioning

confidence: 99%

Cavities determine the pressure unfolding of proteins

Roche

José

Norberto

et al. 2012

350

483

“…The charged and polar groups on the protein surface are already electrostricted and suffer smaller pressure effects than the hydrophobic and less polar groups. A new methodology, pressure perturbation calorimetry (PPC), has made it possible to evaluate the changes in hydration of the side chains as a function of temperature (62)(63)(64). In PPC, the thermal expansion coefficient of a peptide or a protein is obtained directly from the heat absorbed or released after small pressure jumps.…”

Section: Discussionmentioning

confidence: 99%

A hypothesis to reconcile the physical and chemical unfolding of proteins

Oliveira

Silva

2015

High pressure (HP) or urea is commonly used to disturb folding species. Pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the protein-solvent system. However, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though proteinurea interactions are considered to be crucial. Here, we provide NMR spectroscopy and 3D reconstructions from X-ray scattering to develop the "push-and-pull" hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by HP. In studying MpNep2 from Moniliophthora perniciosa, we tracked two cooperative units using HP-NMR as MpNep2 moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. At subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by NMR intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle X-ray scattering (SAXS)]. This state has a higher susceptibility to pressure denaturation (lower p 1/2 and larger ΔV u ); thus, urea and HP share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. These observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes.protein folding | hydrostatic pressure | urea | NMR | SAXS

“…Different solvents can interact differently with the protein and its hydration shell and can change the dynamic and static properties and, in particular, the stability (19,20,(24)(25)(26)(27)(28)(29). Fig.…”

Section: Fractional Viscosity Dependence Of Protein Motionsmentioning

confidence: 99%

Protein folding is slaved to solvent motions

Frauenfelder

Fenimore

Chen

et al. 2006

272

281

Proteins, the workhorses of living systems, are constructed from chains of amino acids, which are synthesized in the cell based on the instructions of the genetic code and then folded into working proteins. The time for folding varies from microseconds to hours. What controls the folding rate is hotly debated. We postulate here that folding has the same temperature dependence as the ␣-fluctuations in the bulk solvent but is much slower. We call this behavior slaving. Slaving has been observed in folded proteins: Large-scale protein motions follow the solvent fluctuations with rate coefficient k␣ but can be slower by a large factor. Slowing occurs because large-scale motions proceed in many small steps, each determined by k␣. If conformational motions of folded proteins are slaved, so a fortiori must be the motions during folding. The unfolded protein makes a Brownian walk in the conformational space to the folded structure, with each step controlled by k␣. Because the number of conformational substates in the unfolded protein is extremely large, the folding rate coefficient, kf, is much smaller than k␣. The slaving model implies that the activation enthalpy of folding is dominated by the solvent, whereas the number of steps nf ‫؍‬ k␣͞kf is controlled by the number of accessible substates in the unfolded protein and the solvent. Proteins, however, undergo not only ␣-but also ␤-fluctuations. These additional fluctuations are local protein motions that are essentially independent of the bulk solvent fluctuations and may be relevant at late stages of folding.folding energy landscape ͉ fractional viscosity dependence ͉ internal viscosity ͉ Maxwell relation ͉ protein-solvent interaction P roteins in cells fold and unfold continuously. Consequently, an understanding of folding rates is key. The distribution and strength of contacts in the native state is one ingredient that influences the rate of folding (1). A second ingredient is the effect of the solvent, because protein motions are intimately linked to the motions of the environment. Our slaving model quantifies the linkage. The model is based on three concepts: Proteins assume a large number of different conformations or substates (2), their organization is described by a hierarchic energy landscape (3), and large-scale protein fluctuations follow the ␣-relaxation (Debye or dielectric relaxation) in the bulk solvent (4-6). Here we propose that these concepts also are valid for folding and that they lead to the model pictured in Fig. 1. Fig. 1a is a cartoon of folding and a 1D cross-section through the high-dimensional energy landscape. Fig. 1b is a 2D cross-section.