2024-03-29T07:05:13Zhttps://eprints.lib.hokudai.ac.jp/dspace-oai/requestoai:eprints.lib.hokudai.ac.jp:2115/766332022-11-17T02:08:08Zhdl_2115_20039hdl_2115_116Uncovering dehydration in cytochrome c refolding from urea- and guanidine hydrochloride-denatured unfolded state by high pressure spectroscopyKonno, ShoheiDoi, KentaroIshimori, Koichirohydrophobic effectpartial molar volume changehemecytochrome c430To investigate the dehydration associated with protein folding, the partial molar volume changes for protein kunfolding (Delta V-u) in cytochrome c (Cyt c) were determined using high pressure absorption spectroscopy. Delta V-u values for the unfolding to urea- and guanidine hydrochloride (GdnHCl)-denatured Cyt c were estimated to be 56 +/- 5 and 29 +/- 1 mL mol(-1), respectively. Considering that the volume change for hydration of hydrophobic groups is positive and that Cyt c has a covalently bonded heme, a positive Delta V-u 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 c than in urea-denatured Cyt c, resulting in the smaller positive Delta V-u. On the other hand, urea is a relatively weak denaturant and urea-denatured Cyt c 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 Delta V-u (4 +/- 2 mL mol(-1)) in urea- denatured Cyt c. 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 c by estimating Delta V-u, which allows us to discuss the hydrated structures in the denatured states of proteins.Biophysical Society of JapanJournal Articlehttp://hdl.handle.net/2115/76633Biophysics and physicobiology1618272020-01-30enginfo:doi/10.2142/biophysico.16.0_18none