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

Abstract

Ribonuclease T 1 (RNase T 1) carboxymethylated at the γ-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 T 1 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 α and δ 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 T 1, the transition curve of the Asn-81 α proton was identical with that of the Ile-90 δ methyl protons, suggesting that the thermal unfolding occurred simultaneously in every part of the molecule of CM-RNase T 1 as well as of intact RNase T 1. The midpoint of unfolding was 52°C for intact RNase T 1, and was increased by 9°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.