Folding and unfolding kinetics of the proline-to-alanine mutants of bovine pancreatic ribonuclease A.


Four single mutants (P42A, P93A, P114A, and P117A) of bovine pancreatic ribonuclease A (RNase A) in which each mutant has one of the four prolines of RNase A changed to alanine were prepared. The physical properties of these four mutants indicate that their native structure is essentially identical to that of wild-type RNase A. The disulfide-intact forms of these proteins were denatured in guanidine hydrochloride (Gdn.HCl) and then refolded by dilution of the Gdn.HCl. Single-jump folding, single-jump unfolding, and double-jump unfolding/folding stopped-flow experiments were carried out on wild-type and the four proline mutants of RNase A using absorption detection to follow the folding kinetics. The single-jump folding experiments carried out at six different final Gdn.HCl concentrations indicate that the folding rate constants of individual steps for the mutants are similar to those of wild-type RNase A. The Tyr92-Pro93 peptide bond has a cis conformation in native wild-type RNase A, and the results from our double-jump stopped-flow experiments indicate that the Tyr92-Ala93 peptide bond in the P93A mutant of RNase A is also cis in the native state. The existence of two cis peptide bonds (preceding Pro93 and Pro114) in wild-type RNase A is probably due to (as-yet-unidentified) long-range interactions, and such interactions are presumably the origin of a cis peptide bond even when alanine is substituted for Pro93. The data from the double-jump stopped-flow experiments are interpreted in terms of a folding/unfolding model. This model specifies the cis/trans isomerization state of the unfolded species (Uvf, Uf, Um, and Us) at each X-Pro peptide bond. Also, this model confirms the existence of several previously postulated chain-folding initiation sites. Study holds ProTherm entries: 3215, 3216, 3217, 3218, 3219 Extra Details: ribonuclease A; proline; alanine; rate constant;,long-range interactions; kinetics

Submission Details

ID: 73sG4gV43

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:21 p.m.

Version: 1

Publication Details
Dodge RW;Scheraga HA,Biochemistry (1996) Folding and unfolding kinetics of the proline-to-alanine mutants of bovine pancreatic ribonuclease A. PMID:8634286
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