title: 1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids.
abstract: Two-dimensional (2D) 1H NMR experiments using deuterium labeling have been carried out to investigate the solution of ribonuclease HI (RNase HI) from Escherichia coli (E. coli), which consists of 155 amino acids. To simplify the 1H NMR spectra, two fully deuterated enzymes bearing several protonated amino acids were prepared from an RNase HI overproducing strain of E. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, Ile, Val, and Leu. The other was labeled by only protonated His and Ile. The 2D 1H NMR spectra of these deuterated RNase HI proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.
authors.affiliation: Protein Engineering Research Institute, Osaka, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Oda
authors.forename: H
authors.initials: H
authors.lastname: Nakamura
authors.forename: T
authors.initials: T
authors.lastname: Yamazaki
authors.forename: K
authors.initials: K
authors.lastname: Nagayama
authors.forename: M
authors.initials: M
authors.lastname: Yoshida
authors.forename: S
authors.initials: S
authors.lastname: Kanaya
authors.forename: M
authors.initials: M
authors.lastname: Ikehara
mesh_terms: D000595:Amino Acid Sequence; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010957:Plasmids; D011487:Protein Conformation; D011522:Protons; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics
chemical_list: D011522:Protons; D006859:Hydrogen; D003903:Deuterium; D016914:Ribonuclease H