Protein stability in mixed solvents: From transfer thermodynamics to the denaturation by urea and back

The conformational equilibrium between folded and unfolded protein structures is sensitive to the presence of cosolvents in the surrounding solution, and several small organic molecules are known to modulate the functioning of proteins in cells by shifting the equilibrium toward folded (stabilizers) or unfolded states (denaturants). The molecular mechanisms behind these effects are not yet fully understood and are a matter of intense research. In the past, successful models have been devised which predict the effects of cosolvents on proteins on the basis of their effects on small model compounds. E. g., in the popular transfer model (TM) the latter are quantified by the free energies of the transfer (TFEs) of the model compounds between water and cosolvent solutions. In this thesis, we examine and discuss the interpretation and measurement of TFEs. Moreover, we deal with the application of TFE-based bottom-up approaches in the study of the denaturing mechanism of urea—the probably most studied and nonetheless most controversially discussed cosolvent. The highlights of this work are: • We present a detailed and comprehensible explanation for the role of the concentration scale in the definition of TFEs and show that only the TFE that is defined in the molarity scale can be interpreted directly in terms of favorable or unfavorable solute-solvent interactions. • We uncover an inconsistency and a compensating error in the nowadays established implementation of the TM. After their revision, the TM predicts that both the protein backbone and the side chains play a role in denaturation by urea. This is in line with many recent studies and thus paves the way toward a unified understanding of urea’s denaturing mechanism. Previous applications of the TM predicted a contrasting mode of action and this discrepancy was considered a major concern. • We present a molecular dynamics study which provides further insight into urea’s denaturing mechanism. It suggests that protein denaturation by urea is the resultant of a complex and subtle interplay of various types of interactions between all solution components, being the protein, urea, and water. • We propose a new measuring method for the determination of TFEs for transfers between pure and mixed solvents and point out that it is necessary to reassess the accuracies of currently employed measuring approaches. First steps into that direction suggest that some currently used measuring methods might not be as accurate as presumed so far