Protein Phase Diagrams II: Nonideal Behavior of Biochemical Reactions in the Presence of Osmolytes

AbstractIn the age of biochemical systems biology, proteomics, and high throughput methods, the thermodynamic quantification of cytoplasmatic reaction networks comes into reach of the current generation of scientists. What is needed to efficiently extract the relevant information from the raw data is a robust tool for evaluating the number and stoichiometry of all observed reactions while providing a good estimate of the thermodynamic parameters that determine the molecular behavior. The recently developed phase-diagram method, strictly speaking a graphical representation of linkage or Maxwell Relations, offers such capabilities. Here, we extend the phase diagram method to nonideal conditions. For the sake of simplicity, we choose as an example a reaction system involving the protein RNase A, its inhibitor CMP, the osmolyte urea, and water. We investigate this system as a function of the concentrations of inhibitor and osmolyte at different temperatures ranging from 280K to 340K. The most interesting finding is that the protein-inhibitor binding equilibrium depends strongly on the urea concentration—by orders-of-magnitude more than expected from urea-protein interaction alone. Moreover, the m-value of ligand binding is strongly concentration-dependent, which is highly unusual. It is concluded that the interaction between small molecules like urea and CMP can significantly contribute to cytoplasmic nonideality. Such a finding is highly significant because of its impact on renal tissue where high concentrations of cosolutes occur regularly

An analysis of the molecular origin of osmolyte-dependent protein stability

Protein solvation is the key determinant for isothermal, concentration-dependent effects on protein equilibria, such as folding. The required solvation information can be extracted from experimental thermodynamic data using Kirkwood-Buff theory. Here we derive and discuss general properties of proteins and osmolytes that are pertinent to their biochemical behavior. We find that hydration depends very little on osmolyte concentration and type. Strong dependencies on both osmolyte concentration and type are found for osmolyte self-solvation and protein–osmolyte solvation changes upon unfolding. However, solvation in osmolyte solutions does not involve complex concentration dependencies as found in organic molecules that are not used as osmolytes in nature. It is argued that the simple solvation behavior of naturally occurring osmolytes is a prerequisite for their usefulness in osmotic regulation in vivo