Folding mechanism of ribonuclease T1 in the absence of the disulfide bonds.


Abstract

In the absence of its two disulfide bonds, ribonuclease T1 can exist in a native-like folded conformation when > or = 2 M NaCl is present. We measured the kinetics of unfolding and refolding of two reduced and carboxymethylated variants of ribonuclease T1 with one cis proline (the Ser54Gly/Pro55Asn variant) and with two cis prolines (the wild-type protein) as a function of the NaCl concentration. Single and double mixing techniques were used. Analysis of the kinetic results demonstrates that the two cis prolyl bonds at Pro39 and Pro55 remain cis in the folded state after the reduction and carboxymethylation of the disulfide bonds. Folded molecules with trans isomers could not be found. The substitution of cis-Pro55 influences the proline-limited folding reaction, and the analysis of the changes in the folding kinetics shows that the trans-->cis isomerizations of both prolines are slow and are rate-determining steps for the refolding of ribonuclease T1 in the presence as well as in the absence of the disulfide bonds. The direct folding reaction of protein chains with correct prolyl isomers is also affected by the Ser54Gly/Pro55Asn mutation. The rate of refolding is decreased, whereas the rate of unfolding is almost unaffected. The kinetic analysis points to two main consequences of the Ser54Gly/Pro55Asn mutation for the stability and the folding mechanism of RNase T1. It is moderately destabilizing, because the deletion of a conformationally restricted residue (Pro55-->Asn) and the insertion of a flexible residue (Ser54-->Gly) both tend to increase the entropy of the unfolded state. The cis<-->trans isomerization of Pro55 is abolished, however, leading to a decrease in the entropy of the unfolded protein. These two entropic contributions seem to partially compensate each other, and the net change in free energy as a consequence of the Ser54Gly/Pro55Asn double mutation is very small. Study holds ProTherm entries: 4527, 4528 Extra Details: measurements were made in the absence of disulfide bonds disulfide bonds; cis prolines; reduction; carboxymethylation;,cis<-->trans isomerization; entropic contributions

Submission Details

ID: JhRDLJPX3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Mücke M;Schmid FX,Biochemistry (1994) Folding mechanism of ribonuclease T1 in the absence of the disulfide bonds. PMID:7981223
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
1B2M 1998-11-27T00:00:00+0000 2.0 THREE-DIMENSIONAL STRUCTURE OF RIBONULCEASE T1 COMPLEXED WITH AN ISOSTERIC PHOSPHONATE ANALOGUE OF GPU: ALTERNATE SUBSTRATE BINDING MODES AND CATALYSIS.
1BIR 1996-01-04T00:00:00+0000 1.8 RIBONUCLEASE T1, PHE 100 TO ALA MUTANT COMPLEXED WITH 2' GMP
1BU4 1998-09-11T00:00:00+0000 1.9 RIBONUCLEASE 1 COMPLEX WITH 2'GMP
1BVI 1998-09-15T00:00:00+0000 1.9 RIBONUCLEASE T1 (WILDTYPE) COMPLEXED WITH 2'GMP
1CH0 1999-03-30T00:00:00+0000 2.3 RNASE T1 VARIANT WITH ALTERED GUANINE BINDING SEGMENT
1DET 1996-02-20T00:00:00+0000 1.8 RIBONUCLEASE T1 CARBOXYMETHYLATED AT GLU 58 IN COMPLEX WITH 2'GMP
1FYS 2000-10-03T00:00:00+0000 2.0 Ribonuclease T1 V16C mutant
1FZU 2000-10-04T00:00:00+0000 1.8 RNAse T1 V78A mutant
1G02 2000-10-05T00:00:00+0000 1.86 Ribonuclease T1 V16S mutant
1GSP 1997-11-28T00:00:00+0000 2.2 RIBONUCLEASE T1 COMPLEXED WITH 2',3'-CGPS, 1 DAY

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Guanyl-specific ribonuclease T1 P00651 RNT1_ASPOR