NMR STRUCTURAL-ANALYSIS OF AN ANALOG OF AN INTERMEDIATE FORMED IN THERATE-DETERMINING STEP OF ONE PATHWAY IN THE OXIDATIVE FOLDING OF BOVINE PANCREATIC RIBONUCLEASE-A - AUTOMATED-ANALYSIS OF H-1, C-13, AND N-15 RESONANCE ASSIGNMENTS FOR WILD-TYPE AND [C65S, C72S] MUTANT FORMS

A three-disulfide intermediate, des-[65-72] RNase A, lacking the disul fide bond between Cys65 and Cys72, is formed in one of the rate-determ ining steps of the oxidative regeneration pathways of bovine pancreati c ribonuclease A (RNase A). An analog of this intermediate, [C65S, C72 S] RNase A, has been characterized in terms of structure and thermodyn amic stability. Triple-resonance NMR data were analyzed using an autom ated assignment program, AUTOASSIGN. Nearly all backbone H-1, C-13, an d N-15 resonances and most side-chain C-13(beta) resonances of both wi ld-type (wt) and [C65S, C72S] RNase A were assigned unambiguously. Ana lysis of NOE, C-13(alpha) chemical shift, and (3)J(H-N-H-alpha) scalar coupling data indicates that the regular backbone structure of the ma jor form of [C65S, C72S] RNase A is very similar to that of the major form of wt RNase A, although small structural differences are indicate d in the mutation site and in spatially adjacent beta-sheet structures comprising the hydrophobic core. Thermodynamic analysis demonstrates that [C65S, C72S] RNase A (T-m of 38.5 degrees C) is significantly les s stable than wt RNase A (T-m of 55.5 degrees C) at pH 4.6. Although t he structural comparison of wt RNase A and this analog of an oxidative folding intermediate indicates only localized effects around the Cys6 5 and Cys72 sites, these thermodynamic measurements indicate that form ation of the fourth disulfide bond, Cys65-Cys72, on this oxidative fol ding pathway results in global stabilization of the native chain fold. This conclusion is supported by comparisons of amide H-1/H-2 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 signi ficantly in stabilizing the structure of the hydrophobic core of the n ative protein. Formation of this disulfide bond in the final step of t his oxidative folding pathway provides significant stabilization of th e native-like structure that is present in the corresponding three-dis ulfide folding intermediate.