Denaturing action of urea and guanidine hydrochloride towards two thermophilic esterases.
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ABSTRACT:
SUBMITTER: Del Vecchio P
PROVIDER: S-EPMC1222942 | biostudies-other | 2002 Nov
REPOSITORIES: biostudies-other
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Project description:To investigate the dehydration associated with protein folding, the partial molar volume changes for protein unfolding (?V u) in cytochrome c (Cyt c) were determined using high pressure absorption spectroscopy. ?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 ?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 ?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 ?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 ?V u, which allows us to discuss the hydrated structures in the denatured states of proteins.
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