Preferential water exclusion in protein unfolding.

Association of water with protein plays a central role in the latter’s folding, structure acquisition, ligand binding, catalytic reactivity, oligomerization, and crystallization. Because these phenomena are also influenced by the net charge content on the protein, the present study examines the association of water with cytochrome c held at different pH values so as to allow its side chains to ionize to variable extents. Equilibrium unfolding of differently charged cytochrome c molecules in water–methanol binary mixtures, where the alcohol acts as the cosolvent denaturant, was used to quantify the preferential exclusion of water during the unfolding transition. The extent of exclusion was found to be related to the net-charge-dependent molecular expansion of the protein in an alcohol-free aqueous medium. The degree of water exclusion was also found to be linearly related to the observed rate of protein unfolding, where the net charge contents of the initial and final states are the same. The results sugge...