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Earlier studies of the unfolding pathway of native bovine pancreatic ribonuclease A (using dithiothreitol as the reducing agent) revealed that the three-disulfide species lacking the disulfide bond between cysteine 65 and cysteine 72 is the most highly populated intermediate [Rothwarf & Scheraga (1991) J. Am. Chem. Soc. 113, 6293-6294]. This unfolding intermediate is referred to as des-[65-72]-RNase A. In order to determine the role of des-[65-72]-RNase A, i.e. of the 65-72 disulfide bond, in the structural folding/unfolding processes of RNase A, the stability and structure of this unfolding intermediate were determined by examining its thermal transition curve and by using two- and three-dimensional homonuclear 1H NMR spectroscopy. The midpoint of the thermal transition of des-[65-72]-RNase A was found to be 17.8 degrees C lower than that of native RNase A. A set of conformations that are consistent with the NMR-derived constraints was obtained by minimizing, first, a variable-target function and, then, the conformational energy. These conformations exhibit a well-defined structure that is very similar to that of native ribonuclease A in regions where the native protein has a regular backbone structure such as a beta-sheet or a helix. Some of the loop regions of the several computed structures exhibit large deviations from each other as well as from native ribonuclease A. However, these results indicate that des-[65-72]-RNase A has a close structural similarity to RNase A in all regions with the only major differences occurring in a loop region comprising residues 60-72. This led to the conclusion that, in reduction pathways that include des-[65-72]-RNase A (at 25 degrees C, pH 8.0), the rate-determining step corresponds to a partial unfolding event in one region of the protein and not to a global conformational unfolding process. The results further suggest that, in the regeneration pathways involving des-[65-72]-RNase A, the loop region from 60 to 72 is the last to fold.

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Conformational analysis of the neurohypophyseal hormones oxytocin (OT) and arginine‐vasopressin (AVP) has been carried out using two different computational approaches and three force fields, namely by the Electrostatically Driven Monte Carlo (EDMC) method, with the Empirical Conformational Energy Program for Peptides (ECEPP/3) force field or with the ECEPP/3 force field plus a hydration‐shell model, and by simulated‐annealing molecular dynamics with the Consistent Valence Force Field (CVFF). The low‐energy conformations obtained for both hormones were classified using the minimal‐tree clustering algorithm and characterized according to the locations of β‐turns in the cyclic moieties. Calculations with the CVFF force field located conformations with a β‐turn at residues 3 and 4 as the lowest energy ones both for OT and for AVP. In the ECEPP/3 force field the lowest energy conformation of OT contained a β‐turn at residues 2 and 3, conformations with this location of the turn being higher in energy for AVP. The later difference can be attributed to the difference in the size of the side chain in position 3 of the sequences: the bulkier phenylalanine residue of AVP in combination with the bulky Tyr2 residue hinders the formation of a turn at residues 2 and 3. Conformations of OT and AVP with a turn at residues 3,4 were in the best agreement with the x‐ray structures of deaminooxytocin and pressinoic acid (the cyclic moiety of vasopressin), respectively, and with the nmr‐derived distance constraints. Generally, the low‐energy conformations obtained with the hydration‐shell model were in a better agreement with the experimental data than the conformations calculated in vacuo. It was found, however, that the obtained low‐energy conformations do not satisfy all of the nmr‐derived distance constraints and the nuclear Overhauser effect pattern observed in nmr studies can be fully explained only by assuming a dynamic equilibrium between conformations with β‐turns at residues 2.3, 3.4, and 4.5. The low‐energy structures of OT with a β‐turn at residues 2.3 have the disulfide ring conformations close to the model proposed recently for a potent bicyclic antagonist of OT [M.D. Shenderovich et al. (1994) Polish Journal of Chemistry, Vol. 25, pp. 921–927], although the native hormone differs from the bicyclic analogue by the conformation of the C‐terminal tripeptide. This finding confirms the hypothesis of different receptor‐bound conformations of agonists and antagonists of OT. © 1996 John Wiley & Sons, Inc.

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