open access

https://hdl.handle.net/2115/76633

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

英語

cytochrome c

2020-01-30

Biophysical Society of Japan

Biophysics and physicobiology

16

18-27

To 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.

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