Delving into controversial dichotomy of direct and indirect mechanisms of Trehalose: In search of unanimous consensus

Abstract

Trehalose, a carbohydrate-based osmolyte, is a widely known protein stabilizer and aggregation inhibitor for numerous proteins. However, the origin of its mechanistic behavior is ambiguous, preventing its therapeutic applicability for broad range of diseases. The controversial reports of direct versus indirect mode of action of trehalose segregates it into two different branches with the potential to fit into both of them. The knowledge of the link connecting them is essential to dissect its placement and comprehensively describe the behavior of trehalose under different environmental conditions and on different proteins. In this regard, we have introspected trehalose’s direct and indirect interaction with residues of varying charge and nature, by simulating five physiologically relevant peptides containing acidic, basic, neutral, hydrophilic and hydrophobic amino acids respectively, and two proteins under different temperature conditions. It was observed that trehalose prefers to favorably bind with side chains of negatively charged acidic amino acid residues, irrespective of force field. The conjecture was validated by mutating these residues with neutral and hydrophobic groups, and was also substantiated by simulating full length proteins. In addition, existing biophysical experimental literature was explained in a different way as per our hypothesis. Ratio of exposed to total surface area of acidic amino acid residues is of prime importance. Trehalose’s perturbation to water molecules viz structural orientation and dynamical diffusion was found to be more in the hydration layer of trehalose. Thus, we propose that the indirect effect is prominent in the neighborhood of trehalose molecules, the distribution of which in turn is guided by direct interactions of trehalose with acidic patches on the protein surface. This way we have bridged the gap between the two hypotheses. We assert that the cause of trehalose’s mechanistic action towards protein stabilization and aggregation resides in its direct interaction with protein.