Revisiting the conundrum of trehalose stabilization.

Protein aggregation and loss of protein's biological functionality are manifestations of protein instability. Cosolvents, in particular trehalose, are widely accepted antidotes against such destabilization. Although numerous theories have been promulgated in the literature with regard to its mechanism of stabilization, the present scenario is still elusive in view of the discrepancies existing in them. To this end, we have revisited the conundrum and attempted to rationalize the mechanism by conducting thorough investigation of the effect of trehalose on the native, partially unfolded and denatured states of protein "Lysozyme" by means of molecular dynamic (MD) simulations under different temperature and concentration regimes. Two-dimensional contour plots along with principal component analysis suggest that trehalose molecules offer on-pathway stabilization unaltering the principal direction of protein's motion, although it slows down protein dynamics so that the protein gets trapped in the homogeneous ensemble of conformations closer to the native state. Free energy landscape reveals higher population of native compared to intermediate and denatured states. Delphi results and calculation of the preferential interaction parameter demonstrate that this relative stabilization of the native state can be ascribed to be the consequence of favourable interactions of trehalose with side chains of certain loci on the protein surface encompassing polar flexible residues. Stability of protein results from the observed difference in binding affinity of trehalose for native and denatured states of protein. Our findings are at variance with the common conception of relative destabilization of the denatured state. Rather, we provide evidence for relative stabilization of the native state. This stabilization is due to interplay of protein-trehalose, water-trehalose, water-water, protein-water and trehalose-trehalose interactions.