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


Abstract

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 PDB Entries

Structure ID Release Date Resolution Structure Title
6QMN 2019-02-07T00:00:00+0000 2.31 Crystal structure of a Ribonuclease A-Onconase chimera
1A2W 1998-01-12T00:00:00+0000 2.1 CRYSTAL STRUCTURE OF A 3D DOMAIN-SWAPPED DIMER OF BOVINE PANCREATIC RIBONUCLEASE A
1A5P 1998-02-17T00:00:00+0000 1.6 C[40,95]A VARIANT OF BOVINE PANCREATIC RIBONUCLEASE A
1A5Q 1998-02-17T00:00:00+0000 2.3 P93A VARIANT OF BOVINE PANCREATIC RIBONUCLEASE A
1AFK 1997-03-08T00:00:00+0000 1.7 CRYSTAL STRUCTURE OF RIBONUCLEASE A IN COMPLEX WITH 5'-DIPHOSPHOADENOSINE-3'-PHOSPHATE
1AFL 1997-03-08T00:00:00+0000 1.7 RIBONUCLEASE A IN COMPLEX WITH 5'-DIPHOSPHOADENOSINE 2'-PHOSPHATE AT 1.7 ANGSTROM RESOLUTION
1AFU 1997-03-14T00:00:00+0000 2.0 STRUCTURE OF RIBONUCLEASE A AT 2.0 ANGSTROMS FROM MONOCLINIC CRYSTALS
1AQP 1997-07-31T00:00:00+0000 2.0 RIBONUCLEASE A COPPER COMPLEX
1B6V 1999-01-18T00:00:00+0000 2.0 CRYSTAL STRUCTURE OF A HYBRID BETWEEN RIBONUCLEASE A AND BOVINE SEMINAL RIBONUCLEASE
1BEL 1995-12-21T00:00:00+0000 1.6 HYDROLASE PHOSPHORIC DIESTER, RNA

Relevant UniProtKB Entries

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