Second virial coefficients as a measure of protein–osmolyte interactions

Abstract

The cytoplasm contains high concentrations of cosolutes. These cosolutes include macromolecules and small organic molecules called osmolytes. However, most biophysical studies of proteins are conducted in dilute solutions. Two broad classes of models have been used to describe the interaction between osmolytes and proteins. One class focuses on excluded volume effects, while the other focuses on binding between the protein and the osmolyte. To better understand protein-smolyte interactions, we have conducted sedimentation equilibrium analytical ultracentrifugation experiments using ferricytochrome c as a model protein.From these experiments, we determined the second virial coefficients for a series of osmolytes. We have interpreted the second virial coefficient as a measure of both excluded volume and protein-osmolyte binding. We conclude that simple models are not sufficient to understand the interactions between osmolytes and proteins. Keywords: Excluded volume; second virial coefficient; cytochrome c; osmolytes; analytical ultracentrifugationThe cellular medium in which proteins must function contains high concentrations of cosolutes. The cosolutes include proteins, nucleic acids, and osmolytes, low molecular weight organic molecules with no net charge. Some organisms change their osmolyte composition in response to environmental stresses. For example, cartilaginous marine fish use trimethylamine N-oxide (TMAO) to counteract high concentrations of urea (Yancey et al. 1982). Here, we present data on the interaction between iso-1-ferricytochrome c and osmolytes.Contrary to cellular conditions, most biophysical studies are conducted in dilute solutions (i.e., in the absence of cosolutes). Therefore, data from these studies may bear little resemblance to the way proteins behave in vivo (Zimmerman and Minton 1993).Many osmolytes stabilize proteins in vivo and in vitro (Hofmeister 1888;Singer and Lindquist 1998;Saunders et al. 2000). However, the stabilization mechanism is poorly understood. In general, two classes of models are used to explain the effects of osmolytes on protein stability (Saunders et al. 2000 and references therein). The first class focuses on the binding between osmolytes and proteins. The other class focuses on excluded volume effects arising from the increased steric repulsions between osmolytes and the protein. The binding models claim that osmolyte-induced stability increases arise from preferential binding of the osmolyte to the native state. The excluded volume models focus on the fact that osmolytes limit the conformational freedom of proteins by driving them to their most compact state, the native state. The decrease in conformational freedom arises from steric repulsions between the protein and the osmolyte.The actual mechanism must be a combination of the two classes, and models based on this combination lead to valuable insight. The fact that osmolytes take up space in solution cannot be denied. The resulting steric repulsion is shown by osmolyte-induced stabilization of the A...

Thermodynamic Perspective on the Dock−Lock Growth Mechanism of Amyloid Fibrils

“…The size of urea and TMAO is σ i =7 Å. The larger collision diameter, compared to molecular volume and partial molar volume estimates of TMAO and urea molecules [31], approximately accounts for the ordered first solvation shell of water surrounding the cosolutes.…”

Soft interactions and crowding

“…Their work focused on correlations between protein-cosolute interactions and changes in water-accessible surface area. Similar studies probed the interactions between the test protein cytochrome c and small cosolutes (Weatherly and Pielak 2001) using sedimentation equilibrium analytical ultracentrifugation. Both studies showed that B 23 decreases in systems where the small molecules interact favorably with the test protein and increases in systems when there is enhanced repulsion.…”

Why Is Trehalose an Exceptional Protein Stabilizer?

“…Based on these results it has been suggested that peptide-water interactions dominate the stabilization of chymotrypsinogen by polyols (53), whereas intensification of hydrophobic interactions dominates the polyol-induced stabilization of lysozyme (54). Recently, studies carried out by Weatherly and Pielak (55) suggest that osmolytes can interact differently with proteins and that simple models are not sufficient to understand protein-osmolyte interactions. The present study involving several proteins varying in their molecular size does indicate the contribution of unfavorable peptide-trehalose interactions in protein stability.…”