Exploring the Piezophilic Behavior of Natural Cosolvent Mixtures

Abstract

Proteins are only marginally stable and are hence very sensitive to environmental conditions, such as high and low temperatures or high hydrostatic pressures. In nature, living organisms are able to compensate for extreme environmental conditions and hence rescue proteins from denaturation by using osmolytes. Organic osmolytes are accumulated under anhydrobiotic, thermal, and pressure stresses. Among those osmolytes are amino acids, polyols and sugars (e.g., glycerol and trehalose), methylamines such as trimethylamine-Noxide (TMAO), and urea. TMAO has been found to enhance protein folding and ligand binding most efficiently. On the other hand, urea, a highly concentrated waste product in mammalian kidneys, is a perturbant. It is also a major organic osmolyte in marine elasmobranch fishes. Interestingly, TMAO has been found to counteract perturbations imposed by urea and hydrostatic pressure in deep-sea animals, most effectively at a 2:1 urea:TMAO ratio. In the deep sea, hydrostatic pressures up to the 1 kbar (100 MPa) range prevail, and living organisms have to cope with such extreme environmental conditions. High hydrostatic pressure generally destabilizes the protein structure, inhibits polymerization of proteins and ligand binding. Interestingly, TMAO has been shown to largely offset these pressure effects. In fact, it was found that the amount of TMAO in the cells of a series of marine organisms increases linearly with the depth of the ocean. For that reason, TMAO is thought to serve as pressure counteractant. The term “piezolyte” has been coined for such kind of cosolute. About the underlying mechanism of stabilization by TMAO at ambient pressure conditions several experimental and theoretical (molecular dynamics simulations) articles have been published in recent years. TMAO is largely excluded from the protein surface and enhances the water structure causing greater organization through more and stronger hydrogen bonding among water molecules. However, the mechanism of this “chemical chaperon” at high hydrostatic pressure (HHP) conditions is still unclear. To yield a deeper understanding of this phenomenon, we determined the intermolecular interaction of dense protein solutions in the absence and presence of cosolvent mixtures of TMAO and urea also under HHP conditions. Small-angle Xray scattering (SAXS) experiments on dense lysozyme solutions have been carried out in the pressure range from 1 bar up to 4 kbar. The SAXS technique accurately monitors structural alterations of the protein solution and yields quantitative information on the state-dependent protein– protein interaction potential. As lysozyme is a highly stable protein, pressure-induced effects will only be attributed to changes in the protein–protein interaction of the native protein and how this is influenced by osmolytes. No pressureinduced unfolding of the protein occurs in the pressure range covered. Complementary thermodynamic data, that is, the temperature of unfolding and the volume change upon unfolding of the protein, were obtained by differential scanning (DSC) and pressure perturbation calorimetry (PPC), respectively. To verify that the protein is folded at all solution conditions studied, SAXS measurements on diluted lysozyme solutions (cP= 10 mgmL ) were carried out in the whole pressure range covered. For diluted protein solutions, the scattering intensity I(q) is proportional to the form factor P(q) (q= (4p/l)sin(V/2) is the wave vector transfer, l the wavelength of the X-rays, and V the scattering angle), which depends on the structure and size of the protein. For dilute lysozyme solutions, the radius of gyration of the particle, Rg, could be determined. We found a constant Rg value of (15.1 0.4) up to 4 kbar, indicating the absence of unfolding even at the highest pressure applied. In the case of concentrated protein solutions, the interaction between the particles gives rise to an additional scattering contribution. This SAXS signal can be described as the product of the form factor and an effective structure factor, which is related to the intermolecular structure factor S(q). To relate the structure factor to the protein–protein interaction potential, statistical mechanical model approaches have to be employed. Here, the mean-spherical approximation (MSA) in combination with the DLVO (Derjaguin– Landau–Verwey–Overbeek) potential V(r) has been used. The pair potential V(r) is given as the sum of a hard sphere potential VHS(r), a repulsive screened Coulomb-like potential VSC(r) and an attractive Yukawian potential VY(r), which is frequently used to describe protein–protein interactions (for details, see the Supporting Information). [*] Y. Zhai, Prof. Dr. R. Winter Fakult t Chemie, TU Dortmund Physikalische Chemie—Biophysikalische Chemie Otto-Hahn Str. 6, 44227 Dortmund (Germany) E-mail: roland.winter@tu-dortmund.de