Effect of osmolytes on the exchange rates of backbone amide protons in proteins

Osmolytes are small organic solutes produced by the cells of all organisms (except halobacteria) in high stress situations (e.g. extremes of salt concentration, high temperature, etc.) to stabilize their macromolecules and so conserve biological activity. They do not interact with the macromolecule directly but act by altering the solvent properties in the cellular environment, and so their presence indirectly modifies the stability of proteins. In this paper we examine the effect of a model osmolyte, glycine, on the stabilization of two proteins, chymotrypsin inhibitor 2 and horse heart cytochrome c. We have used NMR to monitor the effect of this osmolyte on amide hydrogen exchange rates, which allows a probe at discrete points within the protein structure. Hydrogen exchange rates of specific backbone amide protons provide information about the localized structural fluctuations that expose these amides to solvent and allow exchange to take place. We find that the presence of a high concentration of glycine osmolyte has a profound stabilizing effect on the proteins studied, which is accompanied by a large reduction of the exchange rate constants of most slowly exchanging amide protons. The spectra indicate that this arises without significant changes in the three-dimensional structure. However, the effects on individual amide protons within a single protein were not uniform, and a wide variation in the magnitude of the effects was observed. This ranged from no apparent change in the exchange rate, to decreases in the exchange rate constant by over 2 orders of magnitude. The osmolyte appears to alter a number of different processes that contribute to the observed exchange rates, and no simple generalization allows prediction of the extent of stabilization at any individual location. The results are discussed in light of the available structures of the proteins studied.