Comparison of Local and Global Stability of an Analogue of a Disulfide-Folding Intermediate with Those of the Wild-Type Protein in Bovine Pancreatic Ribonuclease A: Identification of Specific Regions of Stable Structure along the Oxidative Folding Pathway

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Abstract
We have identified specific regions of the polypeptide chain of bovine pancreatic ribonuclease A (RNase A) that are critical for stabilizing the oxidative folding intermediate des-[40−95] (with three native disulfide bonds but lacking the fourth native Cys40−Cys95 disulfide bond) in an ensemble of largely disordered three-disulfide precursors (3S ⇔ des-[40−95]). A stable analogue of des-[40−95], viz., [C40A, C95A] RNase A, which contains three out of four native disulfide pairings, was previously found to have a three-dimensional structure very similar to that of the wild-type protein. However, it is determined here from GdnHCl denaturation experiments to have significantly reduced global stability, i.e., = 4.5 kcal /mol at 20 °C and pH 4.6. The local stability of [C40A, C95A] RNase A was also examined using site-specific amide 2H/1H exchange measurements at pD 5.0 to determine the individual unfolding free energy of specific residues under both strongly native (12 °C) and more destabilizing (20 °C) conditions. Comparison of the relative stabilities at specific amide sites of [C40A, C95A] RNase A at both temperatures with the corresponding values for the wild-type protein at 35 °C corroborates previous experimental evidence that unidentified intramolecular contacts in the vicinity of the preferentially formed native one-disulfide (C65−C72) loop are crucial for stabilizing early folding intermediates, leading to des-[40−95]. Moreover, values of for residues at or near the third α-helix, and in part of the second β-sheet of [C40A, C95A] RNase A, indicate that these two regions of regular backbone structure contribute to stabilizing the global chain fold of the des-[40−95] disulfide-folding intermediate in the wild-type protein. More significantly, we have identified numerous specific residues in the first α-helix and the first β-sheet of the protein that are stabilized in the final step of the major oxidative regeneration pathway of RNase A (des-[40−95] ⇒ N).

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