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

Authors

Robert Powers, University of Nebraska - LincolnFollow
G. Marius Clore, Laboratory of Chemical Physics, Building 2 National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda, MD
Ad Bax, Laboratory of Chemical Physics, Building 2 National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda, MD
Daniel S. Garrett, Laboratory of Chemical Physics, Building 2 National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda, MD
Stephen J. Stahl, Protein Expression Laboratory, Building 6B Ofice of the Director, National Institutes of Health Bethesda, MD
Paul T. Wingfield, Protein Expression Laboratory, Building 6B Ofice of the Director, National Institutes of Health Bethesda, MD
Angela M. Gronenborn, Laboratory of Chemical Physics, Building 2 National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda, MD

Document Type

Article

Date of this Version

1991

Citation

J. Mol. Biol. (1991) 221. 1081-1090

Comments

U.S. Government Work

Abstract

The solution structure of the ribonuclease H domain of HIV-l 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 (>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 α-helices and five strands arranged in a mixed parallel/antiparallel β-sheet with a + 1, + 1. -3x. -- 1 x 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.