Thermal stabilization of ribonuclease T1 by carboxymethylation at Glu-58 as revealed by 1H nuclear magnetic resonance spectroscopy.


Abstract

Ribonuclease T1 (RNase T1) carboxymethylated at the gamma-carboxyl group of Glu-58 with iodoacetic acid is known to be completely inactive while it retains an almost full substrate-binding ability. In order to further clarify the effects of the carboxymethylation, the thermal stabilities of intact and Glu-58-carboxymethylated (CM-) RNase T1 were compared by measuring 1H NMR spectra at various temperatures. The transition curves of unfolding were obtained by plotting, as a function of temperature, the peak areas for the alpha and delta protons of Asn-81 and Ile-90, respectively, which are well apart from each other in the three-dimensional structure of the enzyme. For each of intact and CM-RNase T1, the transition curve of the Asn-81 alpha proton was identical with that of the Ile-90 delta methyl protons, suggesting that the thermal unfolding occurred simultaneously in every part of the molecule of CM-RNase T1 as well as of intact RNase T1. The midpoint of unfolding was 52 degrees C for intact RNase T1, and was increased by 9 degrees C upon carboxymethylation at Glu-58. This marked stabilization by carboxymethylation is thought to be due to formation of a salt bridge between the introduced carboxymethyl group and the neighboring guanidium group of Arg-77. Study holds ProTherm entries: 10390 Extra Details: RNase T1; carboxymethylated RNase T1; NMR; thermal stability; unfolding;,transition enthalpy

Submission Details

ID: 5V5a2Xx43

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:40 p.m.

Version: 1

Publication Details
Kojima M;Mizukoshi T;Miyano H;Suzuki E;Tanokura M;Takahashi K,FEBS Lett. (1994) Thermal stabilization of ribonuclease T1 by carboxymethylation at Glu-58 as revealed by 1H nuclear magnetic resonance spectroscopy. PMID:7915996
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