Structural characterization of a three-disulfide intermediate of ribonuclease A involved in both the folding and unfolding pathways.
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.
Talluri
S
S
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301.
Rothwarf
D M
DM
Scheraga
H A
HA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D004220:Disulfides; D012259:Ribonuclease, Pancreatic