Uncovering dehydration in cytochrome refolding from urea- and guanidine hydrochloride-denatured unfolded state by high pressure spectroscopy.

To investigate the dehydration associated with protein folding, the partial molar volume changes for protein unfolding (Δ ) in cytochrome (Cyt ) were determined using high pressure absorption spectroscopy. Δ values for the unfolding to urea- and guanidine hydrochloride (GdnHCl)-denatured Cyt were estimated to be 56±5 and 29±1 mL mol, respectively. Considering that the volume change for hydration of hydrophobic groups is positive and that Cyt has a covalently bonded heme, a positive Δ reflects the primary contribution of the hydration of heme. Because of the marked tendency of guanidium ions to interact with hydrophobic groups, a smaller number of water molecules were hydrated with hydrophobic groups in GdnHCl-denatured Cyt than in urea-denatured Cyt , resulting in the smaller positive Δ . On the other hand, urea is a relatively weak denaturant and urea-denatured Cyt is not completely hydrated, which retains the partially folded structures. To unfold such partial structures, we introduced a mutation near the heme binding site, His26, to Gln, resulting in a negatively shifted Δ (4±2 mL mol) in urea-denatured Cyt . The formation of the more solvated and less structured state in the urea-denatured mutant enhanced hydration to the hydrophilic groups in the unfolding process. Therefore, we confirmed the hydration of amino acid residues in the protein unfolding of Cyt by estimating Δ , which allows us to discuss the hydrated structures in the denatured states of proteins.