Volume, expansivity and isothermal compressibility changes associated with temperature and pressure unfolding of Staphylococcal nuclease

We have characterized the temperature- and pressure-induced unfolding of staphylococcal nuclease (Snase) using high precision densitometric measurements. The changes in the apparent specific volume, expansion coefficient and isothermal compressibility were determined by these measurements. To our knowledge, these are the first measurements of the volume and isothermal compressibility changes of a protein undergoing pressure-induced unfolding. In order to aid in interpreting the temperature and pressure dependence of the apparent specific volume of Snase, we have also carried out differential scanning calorimetry under the solution conditions which are used for the volumetric studies. We have seen that large compensating volume and compressibility effects accompany the temperature and pressure-induced protein unfolding. Measurements of the apparent specific volume and thermal expansion coefficient of Snase at ambient pressure indicate the formation of a pre-transitional, molten globule type of intermediate structure about 10 degrees C below the actual unfolding temperature of the protein. Compared to the folded state, the apparent specific volume of the unfolded protein is about 0.3-0.5 % smaller. In addition, we investigated the pressure dependence of the apparent specific volume of Snase at a number of different temperatures. At 45 degrees C we calculate a decrease in apparent specific volume due to pressure-induced unfolding of -3.3 10(-3) cm(3) g(-1) or -55 cm(3) mol(-1). The threefold increase in compressibility between 40 and 70 MPa reflects a transition to a partially unfolded state, which is consistent with our results obtained for the radius of gyration of the pressure-denatured state of Snase. At the lower temperature of 35 degrees C, a significant increase in compressibility around 30 MPa is indicative of the formation of a pressure-induced molten globule-like intermediate. Changes in the apparent volume, expansion coefficient and isothermal compressibility are discussed in terms of instrinsic, hydrational and thermal contributions accompanying the unfolding transition.     

Probing the contribution of internal cavities to the volume change of protein unfolding under pressure.

The structural origin of the decrease in system volume upon protein denaturation by pressure has remained a puzzle for decades. This negative volume change upon unfolding is assumed to arise globally from more intimate interactions between the polypeptide chain and water, including electrostriction of buried charges that become exposed upon unfolding, hydration of the polypeptide backbone and amino acid side chains and elimination of packing defects and internal void volumes upon unfolding of the chain. However, the relative signs and magnitudes of each of these contributing factors have not been experimentally determined. Our laboratory has probed the fundamental basis for the volume change upon unfolding of staphylococcal nuclease (Snase) using variable solution conditions and point mutants of Snase (Royer CA et al., 1993, Biochemistry 32:5222-5232; Frye KJ et al., 1996, Biochemistry 35:10234-10239). Our prior results indicate that for Snase, neither electrostriction nor polar or nonpolar hydration contributes significantly to the value of the volume change of unfolding. In the present work, we investigate the pressure induced unfolding of three point mutants of Snase in which internal cavity size is altered. The experimentally determined volume changes of unfolding for the mutants suggest that loss of internal void volume upon unfolding represents the major contributing factor to the value of the volume change of Snase unfolding.     

Hydration and protein folding in water and in reverse micelles: compressibility and volume changes

The partial specific volume and adiabatic compressibility of proteins reflect the hydration properties of the solvent-exposed protein surface, as well as changes in conformational states. Reverse micelles, or water-in-oil microemulsions, are protein-sized, optically-clear microassemblies in which hydration can be experimentally controlled. We explore, by densimetry and ultrasound velocimetry, three basic proteins: cytochrome c, lysozyme, and myelin basic protein in reverse micelles made of sodium bis (2-ethylhexyl) sulfosuccinate, water, and isooctane and in aqueous solvents. For comparison, we use beta-lactoglobulin (pI = 5.1) as a reference protein. We examine the partial specific volume and adiabatic compressibility of the proteins at increasing levels of micellar hydration. For the lowest water content compatible with complete solubilization, all proteins display their highest compressibility values, independent of their amino acid sequence and charge. These values lie within the range of empirical intrinsic protein compressibility estimates. In addition, we obtain volumetric data for the transition of myelin basic protein from its initially unfolded state in water free of denaturants, to a folded, compact conformation within the water-controlled microenvironment of reverse micelles. These results disclose yet another aspect of the protein structural properties observed in membrane-mimetic molecular assemblies.     

A molecular dynamics simulation of SNase and its hydration shell at high temperature and high pressure

Temperature- and pressure-induced unfolding of staphylococcal nuclease (SNase) was studied by Royer, Winter et al. using a variety of experimental techniques (SAXS, FT-IR and fluorescence spectroscopy, DSC, PPC, densimetry). For a more detailed understanding of the underlying mechanistic processes of the different unfolding scenarios, we have carried out a series of molecular dynamics (MD) computer simulations on SNase. We investigated the initial changes of the structure of the protein upon application of pressure (up to 5 kbar) and discuss volumetric and structural differences between the native and pressure pre-denatured state. Additionally, we have obtained the compressibility of the protein and hydration water and compare these data with experimental results. As water plays a crucial role in determining the structure, dynamics and function of proteins, we undertook a detailed analysis of the structure of the interfacial water and the protein-solvent H-bond network as well. Moreover, we report here also MD results on the temperature-induced unfolding of SNase. The time evolution of the protein volume and solvent accessible surface area during thermal unfolding have been investigated, and we present a detailed discussion of the temperature-induced unfolding pathway of SNase in terms of secondary and tertiary structural changes.     

The volume and compressibility changes of lysozyme associated with guanidinium chloride and pressure-assisted unfolding.

The apparent specific volumes and isentropic compressibilities of hen egg white lysozyme were measured in aqueous guanidinium chloride solutions at 25 degrees C by means of a vibrational densimeter and a sing-around ultrasonic velocimeter. Little transition attributable to a protein unfolding was detected in the partial specific volume, while the partial specific isentropic compressibility decreased slightly around the transition region. The pressure-assisted unfolding was also investigated in aqueous guanidinium chloride solutions by means of ultraviolet spectroscopy. Assuming a two-state transition model, it was found that the free energy change of unfolding depends almost linearly on pressure and the unfolding reaction is accompanied by a small decrease in volume. The compressibility behavior is in conflict with the notion that a protein structure is almost completely unfolded by guanidinium chloride and most of the amino acid residues in the protein interior are exposed to solvent. These results support the current view that globular proteins have some residual structures even in the unfolded state induced by a strong denaturant.     

Protein stability and dynamics in the pressure-temperature plane

The pressure-temperature stability diagram of proteins and the underlying assumptions of the elliptical shape of the diagram are discussed. Possible extensions, such as aggregation and fibril formation, are considered. An important experimental observation is the extreme pressure stability of the mature fibrils. Molecular origins of the diagram in terms of models of the partial molar volume of a protein focus on cavities and hydration. Changes in thermal expansivity, compressibility and heat capacity in terms of fluctuations of the enthalpy and volume change of the unfolding should also focus on these parameters. It is argued that the study of water-soluble polymers might further our understanding of the stability diagram. Whereas the role of water in protein behaviour is unquestioned, the role of cavities is less clear.     

Effects of chaotropic and kosmotropic cosolvents on the pressure-induced unfolding and denaturation of proteins: an FT-IR study on staphylococcal nuclease

FT-IR spectroscopy was used to study the effects of various chaotropic and kosmotropic cosolvents (glycerol, sucrose, sorbitol, K(2)SO(4), CaCl(2), and urea) on the secondary structure and thermodynamic properties upon unfolding and denaturation of staphylococcal nuclease (Snase). The data show that the different cosolvents have a profound effect on the denaturation pressure and the Gibbs free energy (DeltaG(o)) and volume (DeltaV(o) change of unfolding. Moreover, by analysis of the amide I' infrared bands, conformational changes of the protein upon unfolding in the different cosolvents have been determined. An increase, a reduction, or an independence of the volume change of unfolding is observed, depending on the type of cosolvent, which can at least in part be attributed to the formation of a different unfolded state structure of the protein. The data are compared with the corresponding thermodynamic values of DeltaV(o) for the temperature-induced unfolding process of Snase as obtained by pressure perturbation calorimetry, and significant differences are observed and discussed.     

Temperature and pressure denaturation of chignolin: Folding and unfolding simulation by multibaric-multithermal molecular dynamics method

A multibaric‐multithermal molecular dynamics (MD) simulation of a 10‐residue protein, chignolin, was performed. All‐atom model with the Amber parm99SB force field was used for the protein and the TIP3P model was used for the explicit water molecules. This MD simulation covered wide ranges of temperature between 260 and 560 K and pressure between 0.1 and 600 MPa and sampled many conformations without getting trapped in local‐minimum free‐energy states. Folding events to the native β‐hairpin structure occurred five times and unfolding events were observed four times. As the temperature and/or pressure increases, fraction of folded chignolin decreases. The partial molar enthalpy change ΔH and partial molar volume change ΔV of unfolding were calculated as ΔH = 24.1 ± 4.9 kJ/mol and ΔV = ?5.6 ± 1.5 cm³/mol, respectively. These values agree well with recent experimental results. Illustrating typical local‐minimum free‐energy conformations, folding and unfolding pathways were revealed. When chignolin unfolds from the β‐hairpin structure, only the C terminus or both C and N termini open first. It may undergo an α‐helix or 3₁₀‐helix structure and finally unfolds to the extended structure. Difference of the mechanism between temperature denaturation and pressure denaturation is also discussed. Temperature denaturation is caused by making the protein transferred to a higher entropy state and making it move around more with larger space. The reason for pressure denaturation is that water molecules approach the hydrophobic residues, which are not well hydrated at the folded state, and some hydrophobic contacts are broken. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Protein high-force pulling simulations yield low-force results

All-atom explicit-solvent molecular dynamics simulations are used to pull with extremely large constant force (750-3000 pN) on three small proteins. The introduction of a nondimensional timescale permits direct comparison of unfolding across all forces. A crossover force of approximately 1100 pN divides unfolding dynamics into two regimes. At higher forces, residues sequentially unfold from the pulling end while maintaining the remainder of the protein force-free. Measurements of hydrodynamic viscous stresses are made easy by the high speeds of unfolding. Using an exact low-Reynolds-number scaling, these measurements can be extrapolated to provide, for the first time, an estimate of the hydrodynamic force on low-force unfolding. Below 1100 pN, but surprisingly still at extremely large applied force, intermediate states and cooperative unfoldings as seen at much lower forces are observed. The force-insensitive persistence of these structures indicates that decomposition into unfolded fragments requires a large fluctuation. This finding suggests how proteins are constructed to resist transient high force. The progression of [Formula: see text] helix and [Formula: see text] sheet unfolding is also found to be insensitive to force. The force-insensitivity of key aspects of unfolding opens the possibility that numerical simulations can be accelerated by high applied force while still maintaining critical features of unfolding.     

Exploring the temperature-pressure phase diagram of staphylococcal nuclease

The temperature dependence of the pressure-induced equilibrium unfolding of staphylococcal nuclease (Snase) was determined by fluorescence of the single tryptophan residue, FTIR absorption for the amide I' and tyrosine O-H bands, and small-angle X-ray scattering (SAXS). The results from these three techniques were similar, although the stability as measured by fluorescence was slightly lower than that measured by FTIR and SAXS. The resulting phase diagram exhibits the well-known curvature for heat and cold denaturation of proteins, due to the large decrease in heat capacity upon folding. The volume change for unfolding became less negative with increasing temperatures, consistent with a larger thermal expansivity for the unfolded state than for the folded state. Fluorescence-detected pressure-jump kinetics measurements revealed that the curvature in the phase diagram is due primarily to the rate constant for folding, indicating a loss in heat capacity for the transition state relative to the unfolded state. The similar temperature dependence of the equilibrium and activation volume changes for folding indicates that the thermal expansivities of the folded and transition states are similar. This, along with the fact that the activation volume for folding is positive over the temperature range examined, the nonlinear dependence of the folding rate constant upon temperature implicates significant dehydration in the rate-limiting step for folding of Snase.     

Urea Impedes the Hydrophobic Collapse of Partially Unfolded Proteins

Proteins are denatured in aqueous urea solution. The nature of the molecular driving forces has received substantial attention in the past, whereas the question how urea acts at different phases of unfolding is not yet well understood at the atomic level. In particular, it is unclear whether urea actively attacks folded proteins or instead stabilizes unfolded conformations. Here we investigated the effect of urea at different phases of unfolding by molecular dynamics simulations, and the behavior of partially unfolded states in both aqueous urea solution and in pure water was compared. Whereas the partially unfolded protein in water exhibited hydrophobic collapses as primary refolding events, it remained stable or even underwent further unfolding steps in aqueous urea solution. Further, initial unfolding steps of the folded protein were found not to be triggered by urea, but instead, stabilized. The underlying mechanism of this stabilization is a favorable interaction of urea with transiently exposed, less-polar residues and the protein backbone, thereby impeding back-reactions. Taken together, these results suggest that, quite generally, urea-induced protein unfolding proceeds primarily not by active attack. Rather, thermal fluctuations toward the unfolded state are stabilized and the hydrophobic collapse of partially unfolded proteins toward the native state is impeded. As a result, the equilibrium is shifted toward the unfolded state.     

Effect of hydrostatic pressure on conformational changes of canine milk lysozyme between the native, molten globule, and unfolded states

The effect of pressure on the unfolding of the native (N) and molten globule (MG) state of canine milk lysozyme (CML) was examined using ultraviolet (UV) spectroscopy at pH 4.5 and 2.0, respectively. It appeared that the thermally induced unfolding was promoted by the increase of pressure from atmospheric to 100 MPa, which indicates that both the N and MG states of CML unfolded with the decrease of the partial molar volume change (DeltaV). The volume changes needed for unfolding were estimated from the free energy change vs. pressure plots, and these volume changes became less negative from 20 to 60 degrees C. The DeltaV values at 25 degrees C were obtained for the N-MG (-46 cm3/mol) and MG-unfolded-state (U) transition (-40 cm3/mol). With regards to the MG-U transition, this value is contrastive to that of bovine alpha-lactalbumin (BLA) (0.9 cm3/mol), which is homologous to CML. Previous studies revealed that the MG state of CML was significantly more stable, and closer to the N state in structure, than that of BLA. In contrast to the swollen hydrophobic core of the MG state of BLA, our results suggest that the MG state of CML possesses a tightly packed hydrophobic core into which water molecules cannot penetrate.     

Partial molar volumes and adiabatic compressibilities of unfolded protein states

We determined the partial molar volumes, V degrees , and adiabatic compressibilities, K degrees (S), of N-acetyl amino acids with neutralized carboxyl termini, N-acetyl amino acid amides, and N-acetyl amino acid methylamides between 18 and 55 degrees C. The individual compounds in the three classes have been selected so as to collectively cover the 20 naturally occurring amino acid side chains. We interpret our experimental results in terms of the volumetric contributions and hydration properties of individual amino acid side chains and their constituent atomic groups. We also conducted pH-dependent densimetric and acoustic measurements to determine changes in volume and compressibility accompanying protonation of the aspartic acid, glutamic acid, histidine, lysine, and arginine side chains. We use our resulting data to develop an additive scheme for calculating the partial molar (specific) volume and adiabatic compressibility of fully extended polypeptide chains as a function of pH and temperature. We discuss the differences and similarities between our proposed scheme and the reported additive approaches. We compare our calculated volumetric characteristics of the fully extended conformations of apocytochrome c and apomyoglobin with the experimental values measured in water (for apocytochrome c) or acidic pH (for apomyoglobin). At these respective experimental conditions, the two proteins are unfolded. However, the comparison between the calculated and experimental volumetric characteristics suggests that neither apocytochrome c nor apomyoglobin are fully unfolded and retain a sizeable core of solvent-inaccessible groups.     

Decomposition of protein experimental compressibility into intrinsic and hydration shell contributions

The experimental determination of protein compressibility reflects both the protein intrinsic compressibility and the difference between the compressibility of water in the protein hydration shell and bulk water. We use molecular dynamics simulations to explore the dependence of the isothermal compressibility of the hydration shell surrounding globular proteins on differential contributions from charged, polar, and apolar protein-water interfaces. The compressibility of water in the protein hydration shell is accounted for by a linear combination of contributions from charged, polar, and apolar solvent-accessible surfaces. The results provide a formula for the deconvolution of experimental data into intrinsic and hydration contributions when a protein of known structure is investigated. The physical basis for the model is the variation in water density shown by the surface-specific radial distribution functions of water molecules around globular proteins. The compressibility of water hydrating charged atoms is lower than bulk water compressibility, the compressibility of water hydrating apolar atoms is somewhat larger than bulk water compressibility, and the compressibility of water around polar atoms is about the same as the compressibility of bulk water. We also assess whether hydration water compressibility determined from small compound data can be used to estimate the compressibility of hydration water surrounding proteins. The results, based on an analysis from four dipeptide solutions, indicate that small compound data cannot be used directly to estimate the compressibility of hydration water surrounding proteins.     

Molecular dynamics simulation study on the high-pressure behaviour of an AK16 peptide

While most proteins unfold under high-pressure conditions, some high-pressure experiments suggest that an AK16 peptide forms more helical structures. In order to understand this abnormality, molecular dynamics simulations with the simulated tempering method for the isobaric–isothermal ensemble were performed in a wide pressure range from 0.1 MPa to 1.4 GPa. It was found that the fraction of the folded state decreases once and increases after that with increasing pressure. The partial molar volume change from the folded state to unfolded state increases monotonically from a negative value to a positive value with pressure. The behaviour under high-pressure conditions is consistent with the experimental results. The radius of gyration of highly helical structures decreases with increasing pressure. Moreover, interatomic distances of AK16 become shorter at high pressure than at low pressure. These behaviours indicate that the helical structures are squeezed by high pressure.     

Influence of pressure on the low‐frequency vibrational modes of lysozyme and water: A complementary inelastic neutron scattering and molecular dynamics simulation study

We performed complementary inelastic neutron scattering (INS) experiments and molecular dynamics (MD) simulations to study the influence of pressure on the low‐frequency vibrational modes of lysozyme in aqueous solution in the 1 atm–6 kbar range. Increasing pressure induces a high‐frequency shift of the low‐frequency part (<10 meV = 80 cm^?1) of the vibrational density of states (VDOS), g(ω), of both lysozyme and water that reveals a stiffening of the interactions ascribed to the reduction of the protein and water volumes. Accordingly, high pressures increase the curvature of the free energy profiles of the protein quasiharmonic vibrational modes. Furthermore, the nonlinear influence of pressure on the g(ω) of lysozyme indicates a change of protein dynamics that reflects the nonlinear pressure dependence of the protein compressibility. An analogous dynamical change is observed for water and stems from the distortion of its tetrahedral structure under pressure. Moreover, our study reveals that the structural, dynamical, and vibrational properties of the hydration water of lysozyme are less sensitive to pressure than those of bulk water, thereby evidencing the strong influence of the protein surface on hydration water. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Toward resolution of ambiguity for the unfolded state

The unfolded states in proteins and nucleic acids remain weakly understood despite their importance in folding processes; misfolding diseases (Parkinson's and Alzheimer's); natively unfolded proteins (as many as 30% of eukaryotic proteins, according to Fink); and the study of ribozymes. Research has been hindered by the inability to quantify the residual (native) structure present in an unfolded protein or nucleic acid. Here, a scaling model is proposed to quantify the molar degree of folding and the unfolded state. The model takes a global view of protein structure and can be applied to a number of analytic methods and to simulations. Three examples are given of application to small-angle scattering from pressure-induced unfolding of SNase, from acid-unfolded cytochrome c, and from folding of Azoarcus ribozyme. These examples quantitatively show three characteristic unfolded states for proteins, the statistical nature of a protein folding pathway, and the relationship between extent of folding and chain size during folding for charge-driven folding in RNA.     

The heat capacity of proteins

The heat capacity plays a major role in the determination of the energetics of protein folding and molecular recognition. As such, a better understanding of this thermodynamic parameter and its structural origin will provide new insights for the development of better molecular design strategies. In this paper we have analyzed the absolute heat capacity of proteins in different conformations. The results of these studies indicate that three major terms account for the absolute heat capacity of a protein: (1) one term that depends only on the primary or covalent structure of a protein and contains contributions from vibrational frequencies arising from the stretching and bending modes of each valence bond and internal rotations; (2) a term that contains the contributions of noncovalent interactions arising from secondary and tertiary structure; and (3) a term that contains the contributions of hydration. For a typical globular protein in solution the bulk of the heat capacity at 25°C is given by the covalent structure term (close to 85% of the total). The hydration term contributes about 15 and 40% to the total heat capacity of the native and unfolded states, respectively. The contribution of non-covalent structure to the total heat capacity of the native state is positive but very small and does not amount to more than 3% at 25°C. The change in heat capacity upon unfolding is primarily given by the increase in the hydration term (about 95%) and to a much lesser extent by the loss of noncovalent interactions (up to ～5%). It is demonstrated that a single universal mathematical function can be used to represent the partial molar heat capacity of the native and unfolded states of proteins in solution. This function can be experimentally written in terms of the molecular weight, the polar and apolar solvent accessible surface areas, and the total area buried from the solvent. This unique function accurately predicts the different magnitude and temperature dependences of the heat capacity of both the native and unfolded states, and therefore of the heat capacity changes associated with folding/unfolding transitions. © 1995 Wiley-Liss, Inc.     

Molecular dynamics simulation of proteins under high pressure: Structure,function and thermodynamics

BackgroundMolecular dynamics (MD) simulation is well-recognized as a powerful tool to investigate protein structure, function, and thermodynamics. MD simulation is also used to investigate high pressure effects on proteins. For conducting better MD simulation under high pressure, the main issues to be addressed are: (i) protein force fields and water models were originally developed to reproduce experimental properties obtained at ambient pressure; and (ii) the timescale to observe the pressure effect is often much longer than that of conventional MD simulations.Scope of reviewFirst, we describe recent developments in MD simulation methodologies for studying the high-pressure structure and dynamics of protein molecules. These developments include force fields for proteins and water molecules, and enhanced simulation techniques. Then, we summarize recent studies of MD simulations of proteins in water under high pressure.Major conclusionsRecent MD simulations of proteins in solution under pressure have reproduced various phenomena identified by experiments using high pressure, such as hydration, water penetration, conformational change, helix stabilization, and molecular stiffening.General significanceMD simulations demonstrate differences in the properties of proteins and water molecules between ambient and high-pressure conditions. Comparing the results obtained by MD calculations with those obtained experimentally could reveal the mechanism by which biological molecular machines work well in collaboration with water molecules.     

Characterization of low-lying excited states of proteins by high-pressure NMR

Hydrostatic pressure alters the free energy of proteins by a few kJ mol⁻¹, with the amount depending on their partial molar volumes. Because the folded ground state of a protein contains cavities, it is always a state of large partial molar volume. Therefore pressure always destabilises the ground state and increases the population of partially and completely unfolded states. This is a mild and reversible conformational change, which allows the study of excited states under thermodynamic equilibrium conditions. Many of the excited states studied in this way are functionally relevant; they also seem to be very similar to kinetic folding intermediates, thus suggesting that evolution has made use of the ‘natural’ dynamic energy landscape of the protein fold and sculpted it to optimise function. This includes features such as ligand binding, structural change during the catalytic cycle, and dynamic allostery.