Molecular Determinants of Expansivity of Native Globular Proteins: A Pressure Perturbation Calorimetry Study

Abstract

There is a growing interest in understanding how hydrostatic pressure (P) impacts the thermodynamic stability (ΔG) of globular proteins. The pressure dependence of stability is defined by the change in volume upon denaturation, ΔV = (∂ΔG/∂P)T. The temperature dependence of change in volume upon denaturation itself is defined by the changes in thermal expansivity (ΔE), ΔE = (∂ΔV/∂T)P. The pressure perturbation calorimetry (PPC) allows direct experimental measurement of the thermal expansion coefficient, α = E/V, of a protein in the native, αN(T), and unfolded, αU(T), states as a function of temperature. We have shown previously that αU(T) is a nonlinear function of temperature but can be predicted well from the amino acid sequence using α(T) values for individual amino acids (J. Phys. Chem. B 2010, 114, 16166-16170). In this work, we report PPC results on a diverse set of nine proteins and discuss molecular factors that can potentially influence the thermal expansion coefficient, αN(T), and the thermal expansivity, EN(T), of proteins in the native state. Direct experimental measurements by PPC show that αN(T) and EN(T) functions vary significantly for different proteins. Using comparative analysis and site-directed mutagenesis, we have eliminated the role of various structural or thermodynamic properties of these proteins such as the number of amino acid residues, secondary structure content, packing density, electrostriction, dynamics, or thermostability. We have also shown that αN(T) and EN,sp(T) functions for a given protein are rather insensitive to the small changes in the amino acid sequence, suggesting that αN(T) and EN(T) functions might be defined by a topology of a given protein fold. This conclusion is supported by the similarity of αN(T) and EN(T) functions for six resurrected ancestral thioredoxins that vary in sequence but have very similar tertiary structure.

Probing volumetric properties of biomolecular systems by pressure perturbation calorimetry (PPC) – The effects of hydration, cosolvents and crowding

“…Interestingly, the magnitude of the negative expansivities for the nonpolar amino acid side chains is not proportional to their hydrophobicity, but correlates with the size of side chain [1]. Formerly, it was believed that proteins owing relatively high numbers of hydrophilic side chains would cause higher a-values [2], but recent studies using mutations of the surface groups of proteins indicate that the a-values of these proteins were not significantly affected [7,8]. In contrast to the native state of proteins, expansivities of unfolded proteins are more strongly dependent on the nature of the exposed atomic groups and can be empirically estimated based on the contributions of the individual side chains [9].…”

“…A similar PPC study on various genetically engineered mutant proteins supported the exclusion of other possible contributing factors to the expansivity of the native proteins, such as the amount of secondary structural elements, the packing density, dynamics and surface electrostriction. Based on these results, it has been hypothesized that the intrinsic network of interactions that defines the tertiary fold is largely responsible for the native protein's expansivity values and its temperature dependence [8]. Further studies are needed to confirm these findings and to clarify the possible correlation between protein topology and expansion of the folded state as a function of temperature, however.…”

“…This calorimeter has basically the same architecture as a power-compensation differential scanning calorimeter (DSC), but measures the heat flow into or out of a sample upon small isothermal pressure changes (of typically ±5 bar). The method can be applied to study the coefficient of thermal expansion, a, and relative volume changes, DV/V, of aqueous solutions and dispersions of chemical and biochemical systems, including amino acids, proteins, DNA and phospholipid vesicles, micellar systems, and polymers [1][2][3][4][5][6][7][8][9][10][11].…”

Applications of pressure perturbation calorimetry to study factors contributing to the volume changes upon protein unfolding

“…Similarly, the thermal expansion coefficient for the native state a N (T) also shows nonlinear dependence on temperature, although this dependence is not as steep as for the unfolded state, particularly in the temperature range between 0°C and ~60°C (see ref [38] and case studies below).…”

“…The second important factor is that the amino acid substitutions have little effect on the α N (T). In an extensive study of over nine different proteins and several of their variants, we have shown that α N (T) appears to be defined by the topology of a given protein fold [38]. Thus one can use the estimated α N (T) of a more stable variant to fit the data of a less stable one.…”