title: 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.
authors.affiliation: Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Kojima
authors.forename: T
authors.initials: T
authors.lastname: Mizukoshi
authors.forename: H
authors.initials: H
authors.lastname: Miyano
authors.forename: E
authors.initials: E
authors.lastname: Suzuki
authors.forename: M
authors.initials: M
authors.lastname: Tanokura
authors.forename: K
authors.initials: K
authors.lastname: Takahashi
mesh_terms: D004795:Enzyme Stability; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D011487:Protein Conformation; D011522:Protons; D006163:Ribonuclease T1 / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D005971:Glutamates; D011522:Protons; D018698:Glutamic Acid; D006163:Ribonuclease T1