LAUSR

Abstract Obtaining qualitative and quantitative insights into counteraction mechanism of deleterious effects of denaturants on proteins is crucial because of its fundamental impact on biological and industrial processes. In this work, we have employed a combination of ultrasensitive isothermal titration calorimetry and differential scanning calorimetry in understanding the mechanistic aspects of scarcely studied counteraction of denaturing action of guanidine hydrochloride by citrulline, betaine and their combination. The results have enabled a quantitative evaluation of stabilization provided by these biologically important molecules along with insights into the counteraction mechanism on the basis of energetics and strength of interactions. Citrulline is able to provide better stabilization to the protein under stressed environment of guanidine hydrochloride than betaine which stabilizes by a relatively different mechanism. The results suggest that along with weak polar interactions between these molecules and guanidine hydrochloride which tend to pull back the denaturant from the protein surface, preferential exclusion also plays a major role. This conclusion differs from the suggested mode of counteraction of urea by osmolytes based mostly on theoretical considerations which emphasize on engagement of osmolytes with urea, though experimental evidences are still lacking for the same.