Macromolecular crowding and preferential exclusion counteract the effect of protein denaturant: Biophysical aspects

[Display omitted] •Counteracting effect of osmolytes and crowders on protein denaturation studied.•Four osmolytes in combination trigger complete stabilization of denatured protein.•CD spectroscopy indicated unaltered tertiary structure of lysozyme with additives.•Calorimetry indicates insignificant direct interactions of denaturant with crowders.•Lysozyme destabilization by PEG-600 occurs due to hydrophobic interactions. Biological macromolecules like nucleic acids, proteins, lipids, carbohydrates, ribosomes etc. function under intracellular crowded environment. The crowded environment of the cell alters the structural, functional and thermodynamic properties of the macromolecules in contrast to the dilute buffer conditions which are commonly used in experimental studies. In this work, we examine the effect of molecular crowding and preferential exclusion on stability of a model protein, lysozyme. The work was performed using a combination of spectroscopic and calorimetric approach. We employed guanidinium hydrochloride as protein denaturant, molecular crowders PEG 600, dextran 40 and osmolytes glycine, proline, sorbitol, sarcosine individually and in mixture. Our results provide quantitative assessment of stabilization offered by these molecules and mechanistic insights into counteraction mechanism. PEG 600 itself destabilizes the protein while sorbitol does not exert any effect. All other stabilizing agents studied here offered a partial counteraction of denaturation. Our results suggest that chemical interactions of the stabilizing agents with the denaturants play a minimal role in prevention or reversal of denaturation while, favorable interactions with the protein and preferential exclusion are the main cause for protein stability offered.