Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds.


Abstract

Ribonuclease T1 has two disulfide bonds linking cysteine residues 2-10 and 6-103. We have prepared a derivative of ribonuclease T1 in which one disulfide bond is broken and the cysteine residues carboxymethylated, (2-10)-RCM-T1, and three derivatives in which both disulfides are broken and the cysteine residues reduced, R-T1, carboxamidomethylated, RCAM-T1, or carboxymethylated, RCM-T1. The RNA hydrolyzing activity of these proteins has been measured, and urea and thermal denaturation studies have been used to determine conformational stability. The activity, melting temperature, and conformational stability of the proteins are: ribonuclease T1 (100%, 59.3 degrees C, 10.2 kcal/mol), (2-10)-RCM-T1 (86%, 53.3 degrees C, 6.8 kcal/mol), R-T1 (53%, 27.2 degrees C, 3.0 kcal/mol), RCAM-T1 (43%, 21.2 degrees C, 1.5 kcal/mol), and RCM-T1 (35%, 16.6 degrees C, 0.9 kcal/mol). Thus, the conformational stability is decreased by 3.4 kcal/mol when one disulfide bond is broken and by 7.2-9.3 kcal/mol when both disulfide bonds are broken. It is quite remarkable that RNase T1 can fold and function with both disulfide bonds broken and the cysteine residues carboxymethylated. The large decrease in the stability is due mainly to an increase in the conformational entropy of the unfolded protein which results when the constraints of the disulfide bonds on the flexibility are removed. We propose a new equation for predicting the effect of a cross-link on the conformational entropy of a protein: delta Sconf = -2.1 - (3/2)R 1n n, where n is the number of residues between the side chains which are cross-linked. This equation gives much better agreement with experimental results than other forms of this equation which have been used previously. Study holds ProTherm entries: 2271, 2272 Extra Details: Ribonuclease T1; disulfide bond; conformational stability; activity;,melting temperature; conformational entropy

Submission Details

ID: 25puCftS4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:18 p.m.

Version: 1

Publication Details
Pace CN;Grimsley GR;Thomson JA;Barnett BJ,J. Biol. Chem. (1988) Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds. PMID:2457027
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
1BVI 1998-09-15T00:00:00+0000 1.9 RIBONUCLEASE T1 (WILDTYPE) COMPLEXED WITH 2'GMP
8RNT 1991-09-23T00:00:00+0000 1.8 STRUCTURE OF RIBONUCLEASE T1 COMPLEXED WITH ZINC(II) AT 1.8 ANGSTROMS RESOLUTION: A ZN2+.6H2O.CARBOXYLATE CLATHRATE
4HOH 1998-09-14T00:00:00+0000 2.05 RIBONUCLEASE T1 (THR93ALA MUTANT) COMPLEXED WITH 2'GMP
3GSP 1997-12-02T00:00:00+0000 1.9 RIBONUCLEASE T1 COMPLEXED WITH 2',3'-CGPS + 3'-GMP, 4 DAYS
1I0V 2001-01-30T00:00:00+0000 1.23 Ribonuclease T1 in complex with 2'GMP (form I crystal)
3BU4 1998-09-14T00:00:00+0000 1.77 RIBONUCLEASE T1 COMPLEX WITH 2'GMP
5GSP 1997-12-09T00:00:00+0000 1.8 RIBONUCLEASE T1/3'-GMP, 9 WEEKS
4BIR 1998-01-13T00:00:00+0000 1.7 RIBONUCLEASE T1: FREE HIS92GLN MUTANT
1I0X 2001-01-30T00:00:00+0000 1.65 RIBONUCLEASE T1 IN COMPLEX WITH 2'GMP (FORM II CRYSTAL)
5RNT 1991-03-28T00:00:00+0000 3.2 X-RAY ANALYSIS OF CUBIC CRYSTALS OF THE COMPLEX FORMED BETWEEN RIBONUCLEASE T1 AND GUANOSINE-3',5'-BISPHOSPHATE

Relevant UniProtKB Entries

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