Dissimilarity in the Reductive Unfolding Pathways of Two Ribonuclease Homologues

• Published in: Journal of molecular biology 2004-05, Vol.338 (4), p.795-809
• Main Authors: Narayan, Mahesh, Xu, Guoqiang, Ripoll, Daniel R, Zhai, Huili, Breuker, Kathrin, Wanjalla, Celestine, Leung, Howard J, Navon, Amiel, Welker, Ervin, McLafferty, Fred W, Scheraga, Harold A
• Format: Article
• Language: English
• Subjects: Animals; Anura; Cattle; Cysteine - chemistry; Disulfides - chemistry; exposed surface area; Freshwater; global unfolding; in silico mutation; local unfolding; Models, Molecular; Oxidation-Reduction; Protein Conformation; Protein Denaturation; Rana pipiens; reductive unfolding; Ribonuclease, Pancreatic - chemistry; Ribonuclease, Pancreatic - genetics; Ribonucleases - chemistry; Ribonucleases - genetics
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2004.03.014

Using DTT red as the reducing agent, the kinetics of the reductive unfolding of onconase, a frog ribonuclease, has been examined. An intermediate containing three disulfides, Ir, that is formed rapidly in the reductive pathway, is more resistant to further reduction than the parent molecule, indicating that the remaining disulfides in onconase are less accessible to DTT red. Disulfide-bond mapping of Ir indicated that it is a single species lacking the (30–75) disulfide bond. The reductive unfolding pattern of onconase is consistent with an analysis of the exposed surface area of the cysteine sulfur atoms in the (30–75) disulfide bond, which reveals that these atoms are about four- and sevenfold, respectively, more exposed than those in the next two maximally exposed disulfides. By contrast, in the reductive unfolding of the homologue, RNase A, there are two intermediates, arising from the reduction of the (40–95) and (65–72) disulfide bonds, which takes place in parallel, and on a much longer time-scale, compared to the initial reduction of onconase; this behavior is consistent with the almost equally exposed surface areas of the cysteine sulfur atoms that form the (40–95) and (65–72) disulfide bonds in RNase A and the fourfold more exposed cysteine sulfur atoms of the (30–75) disulfide bond in onconase. Analysis and in silico mutation of the residues around the (40–95) disulfide bond in RNase A, which is analogous to the (30–75) disulfide bond of onconase, reveal that the side-chain of tyrosine 92 of RNase A, a highly conserved residue among mammalian pancreatic ribonucleases, lies atop the (40–95) disulfide bond, resulting in a shielding of the corresponding sulfur atoms from the solvent; such burial of the (30–75) sulfur atoms is absent from onconase, due to the replacement of Tyr92 by Arg73, which is situated away from the (30–75) disulfide bond and into the solvent, resulting in the large exposed surface-area of the cysteine sulfur atoms forming this bond. Removal of Tyr92 from RNase A resulted in the relatively rapid reduction of the mutant to form a single intermediate (des [40–95] Y92A), i.e. it resulted in an onconase-like reductive unfolding behavior. The reduction of the P93A mutant of RNase A proceeds through a single intermediate, the des [40–95] P93A species, as in onconase. Although mutation of Pro93 to Ala does not increase the exposed surface area of the (40–95) cysteine sulfur atoms, structural analysis of the mutant reveals that there i