title: Secondary structure of the ribonuclease H domain of the human immunodeficiency virus reverse transcriptase in solution using three-dimensional double and triple resonance heteronuclear magnetic resonance spectroscopy.
abstract: The solution structure of the ribonuclease H domain of HIV-1 reverse transcriptase has been investigated by three-dimensional double and triple resonance heteronuclear magnetic resonance spectroscopy. The domain studied has 138 residues and comprises residues 427 to 560 of the 66 kDa reverse transcriptase with an additional four residues at the N terminus. Initial studies on the wild-type protein were hindered by severe differential line broadening, presumably due to conformational averaging. Mutation of the single tryptophan residue located in a loop at position 113 (position 535 in the reverse transcriptase sequence) to an alanine resulted in much improved spectral properties with no apparent change in structure. 1H, 15N and 13C backbone resonances were assigned sequentially using a range of three-dimensional double and triple resonance heteronuclear experiments on samples of uniformly (greater than 95%) 15N and 15N/13C-labeled protein, and the secondary structure was elucidated from a qualitative analysis of data derived from three-dimensional 15N- and 13C-edited nuclear Overhauser enhancement spectra. The secondary structure comprises three alpha-helices and five strands arranged in a mixed parallel/antiparallel beta-sheet with a +1, +1, -3x, -1x topology. The C-terminal region from residue 114 onwards appears to be conformationally disordered in solution as evidenced by an almost complete absence of sequential and medium range nuclear Overhauser effects.
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
authors.forename: R
authors.initials: R
authors.lastname: Powers
authors.forename: G M
authors.initials: GM
authors.lastname: Clore
authors.forename: A
authors.initials: A
authors.lastname: Bax
authors.forename: D S
authors.initials: DS
authors.lastname: Garrett
authors.forename: S J
authors.initials: SJ
authors.lastname: Stahl
authors.forename: P T
authors.initials: PT
authors.lastname: Wingfield
authors.forename: A M
authors.initials: AM
authors.lastname: Gronenborn
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D015497:HIV-1 / Q000737:chemistry / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D016914:Ribonuclease H / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H