NMR Structural Analysis of an Analog of an Intermediate Formed in the Rate-Determining Step of One Pathway in the Oxidative Folding of Bovine Pancreatic Ribonuclease A: Automated Analysis of 1H, 13C, and 15N Resonance Assignments for Wild-Type and [C65S, C72S] Mutant Forms

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Abstract
A three-disulfide intermediate, des-[65-72] RNase A, lacking the disulfide bond between Cys65 and Cys72, is formed in one of the rate-determining steps of the oxidative regeneration pathways of bovine pancreatic ribonuclease A (RNase A). An analog of this intermediate, [C65S, C72S] RNase A, has been characterized in terms of structure and thermodynamic stability. Triple-resonance NMR data were analyzed using an automated assignment program, AUTOASSIGN. Nearly all backbone 1H, 13C, and 15N resonances and most side-chain 13C(beta) resonances of both wild-type (wt) and [C65S, C72S] RNase A were assigned unambiguously. Analysis of NOE, 13C(alpha) chemical shift, and 3J(H(N)-H(alpha)) scalar coupling data indicates that the regular backbone structure of the major form of [C65S, C72S] RNase A is very similar to that of the major form of wt RNase A, although small structural differences are indicated in the mutation site and in spatially adjacent beta-sheet structures comprising the hydrophobic core. Thermodynamic analysis demonstrates that [C65S, C72S] RNase A (Tm of 38.5 degrees C) is significantly less stable than wt RNase A (Tm of 55.5 degrees C) at pH 4.6. Although the structural comparison of wt RNase A and this analog of an oxidative folding intermediate indicates only localized effects around the Cys65 and Cys72 sites, these thermodynamic measurements indicate that formation of the fourth disulfide bond, Cys65-Cys72, on this oxidative folding pathway results in global stabilization of the native chain fold. This conclusion is supported by comparisons of amide 1H/2H exchange rates which are significantly faster throughout the entire structure of [C65S, C72S] RNase A than in wt RNase A. More generally, our study indicates that the C65-C72 disulfide bond of RNase A contributes significantly in stabilizing the structure of the hydrophobic core of the native protein. Formation of this disulfide bond in the final step of this oxidative folding pathway provides significant stabilization of the native-like structure that is present in the corresponding three-disulfide folding intermediate.