EFFECT OF OSMOLYTES ON THE EXCHANGE-RATES OF BACKBONE AMIDE PROTONS IN PROTEINS

Osmolytes are small organic solutes produced by the cells of all organ isms (except halobacteria) in high stress situations (e.g. extremes of salt concentration, high temperature, etc.) to stabilize their macrom olecules and so conserve biological activity. They do not interact wit h the macromolecule directly but act by altering the solvent propertie s in the cellular environment, and so their presence indirectly modifi es the stability of proteins. In this paper we examine the effect of a model osmolyte, glycine, on the stabilization of two proteins, chymot rypsin inhibitor 2 and horse heart cytochrome c. We have used NMR to m onitor the effect of this osmolyte on amide hydrogen exchange rates, w hich allows a probe at discrete points within the protein structure. H ydrogen exchange rates of specific backbone amide protons provide info rmation about the localized structural fluctuations that expose these amides to solvent and allow exchange to take place. We find that the p resence of a high concentration of glycine osmolyte has a profound sta bilizing effect on the proteins studied, which is accompanied by a lar ge reduction of the exchange rate constants of most slowly exchanging amide protons. The spectra indicate that this arises without significa nt changes in the three-dimensional structure. However, the effects on individual amide protons within a single protein were not uniform, an d a wide variation in the magnitude of the effects was observed. This ranged from no apparent change in the exchange rate, to decreases in t he exchange rate constant by over 2 orders of magnitude. The osmolyte appears to alter a number of different processes that contribute to th e observed exchange rates, and no simple generalization allows predict ion of the extent of stabilization at any individual location. The res ults are discussed in light of the available structures of the protein s studied.