Effect of Osmolytes on Protein Stability and Folding
by Avery, Andrew H.; Rajalingam, Dakshinamurthy; Kumar, T. K. S.
Cells exhibit cellular "coping mechanisms" when faced with osmotic stresses by importing or producing org. compds. called osmolytes which aid in osmotic regulation. Proline is an example of such compds. The primary function of osmolytes is to combat the effects of dehydration in the cell. The stabilization of proteins, which are particularly susceptible to osmotic stresses and are of key importance to the cell's health, is a function of osmolytes. Osmolytes have been shown to directly impact the stability and soly. of proteins, and it has been shown that org. osmolytes aid in protein folding and refolding (as well as the regaining of biol. function) and in preventing protein aggregation. The mode by which osmolytes aid in assuring protein stability is believed to be a solvent-oriented process by which protein folding is facilitated by the preferential ordering of solvent mols., but the exact mechanism of stabilization remains elusive. We characterized the supramol. structure of proline at high concn. in soln. using multi-dimensional NMR spectroscopy and DLS. Results from thermal denaturation studies monitored by steady state fluorescence and DSC indicates that the Tm of the protein increases in the presence of increasing concns. of proline by 20°C, suggesting that thermodn. stability of the protein is enhanced by proline. Further denaturation studies are carried out to analyze other osmolytes, as well. 1H-15N- HSQC two-dimensional NMR expts. were used to reveal the binding sites of proline FGF-1. The results of this study provide useful insights on the mol. mechanism of proline.