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
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