Pressure Perturbation Calorimetry: A New Technique Provides Surprising Results on the Effects of Co‐solvents on Protein Solvation and Unfolding Behaviour

Abstract

We used pressure perturbation calorimetry (PPC), a relatively new and efficient technique, to study the solvation and volumetric properties of proteins in their native and unfolded states. In PPC, the coefficient of thermal expansion of the partial volume of the protein is deduced from the heat consumed or produced after small isothermal pressure jumps (e.g., AE 5 bar), and it strongly depends on the interaction of the protein with the solvent or co-solvent at the protein-solvent interface. Furthermore, the effects of various chaotropic and kosmotropic co-solvents (glycerol, sorbitol, sucrose, urea, guanidinium hydrochloride, guanidinium sulfate, potassium sulfate) on the volume and expansivity changes were measured over a wide concentration range with high precision. Depending on the type of co-solvent and its molar concentration, specific differences were found for the solvation properties and unfolding behaviour of the proteins, and the volume change upon unfolding may even change sign. The aim of the PPC study described herein was two-fold: to obtain more insight into the basic thermodynamic properties of protein solvation and the volume effects accompanying unfolding scenarios of proteins in various co-solvents-as these form the basis for understanding their physiological functions and their use in drug design and formulation-and, more generally, to initiate further valuable applications in studies of other biomolecular and chemical systems.The stability and folding of proteins has fascinated protein chemists for many years, and gaining an understanding of this still remains one of the most challenging goals in the field. In general, a protein's stability depends on the temperature and pressure, its hydration capacity and on the solvent's properties. [1][2][3][4][5][6][7][8][9] An important factor contributing to their stability is also their relative affinity towards a particular reagent (in the present context, a co-solvent) in comparison to water or a buffer solution. In fact, protein studies are conducted almost exclusively in rather complex aqueous solutions. It is also wellknown that the cytoplasm of a cell is relatively crowded, and surface-to-surface gaps of neighbouring organelles and biopolymers are, on average, less than 5 nm. [10,11] Given such proximity, we can expect the structure and dynamics of the solvent to be largely determined by the properties of the biomolecular surfaces, and-vice versa-the surface properties of the bio-

Cavities determine the pressure unfolding of proteins

“…Second, in this and in previous studies (20,21) we find that pressure unfolding profiles shift to higher or lower pressures with changes in solution composition, but the volume changes remain constant, regardless of the pressure range over which the unfolding occurs. Moreover, measurements with PPC (22,23), which involve pressure changes of only 5 bar, reveal volume changes upon unfolding consistent with those measured using spectroscopic approaches.…”

Kinetic, Thermodynamic, and Mechanistic Patterns for Free (Unbound) Cytochrome c at Au/SAM Junctions: Impact of Electronic Coupling, Hydrostatic Pressure, and Stabilizing/Denaturing Additives

“…Our calorimetric data indeed show the stabilization effect at all glucose concentrations applied (Figure 5a). In general, we observed splitting of melting endothermic peaks in two components, indicative of two different mechanisms of protein stabilization by glucose (as also discussed in the literature [59] ). We define the two mechanisms of stabilization as mechanisms I and II as to refer to low-and high-temperature peaks observed.…”

Protein–Protein Interactions in Complex Cosolvent Solutions

“…A similar conclusion has been drawn from thermodynamic measurements on RNase A. [32] Measurements were also performed for a concentration of 1 m sucrose. The corresponding J value of the attractive interaction potential is 2.5 k B T for the 4 wt % solution and 1.25 k B T for the 10 wt % lysozyme solution, with d values of about 9.25 for the 4 wt % and 16.25 for the 10 wt % solution.…”

“…As an effective natural osmolyte, sucrose is expected to have a similar effect on the intermolecular interaction of proteins, although a different concentration dependence of the hydration properties has been observed in thermodynamic measurements by Ravindra and Winter. [32] The dielectric permittivities of the solutions with 500 mm and 1 m sucrose solutions were determined to be 75.4 and 73.1, respectively. The scattering patterns for different lysozyme concentrations in the presence of 500 mm sucrose (data not shown) exhibit a stronger intermolecular correlation peak with lower intensities at low Q values.…”