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. Study holds ProTherm entries: 4518 Extra Details: unfolding pathway; disulfide bond; conformational energy;,beta-sheet; helix; rate-determining step

Submission Details

ID: inPmkk9z

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:25 p.m.

Version: 1

Publication Details
Talluri S;Rothwarf DM;Scheraga HA,Biochemistry (1994) Structural characterization of a three-disulfide intermediate of ribonuclease A involved in both the folding and unfolding pathways. PMID:8068682
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Ribonuclease pancreatic P61824 RNAS1_BISBI
100.0 Ribonuclease pancreatic P61823 RNAS1_BOVIN
96.8 Ribonuclease pancreatic P67926 RNAS1_CAPHI
96.8 Ribonuclease pancreatic P67927 RNAS1_SHEEP
95.2 Ribonuclease pancreatic P00657 RNAS1_BUBBU
96.0 Ribonuclease pancreatic P07847 RNAS1_AEPME
93.5 Ribonuclease pancreatic P07848 RNAS1_EUDTH
95.2 Ribonuclease pancreatic P00660 RNAS1_CONTA
92.7 Ribonuclease pancreatic P00668 RNAS1_ANTAM
90.3 Ribonuclease pancreatic P00662 RNAS1_GIRCA
96.0 Ribonuclease pancreatic Q29606 RNAS1_ORYLE