Analysis of the Backbone Dynamics of the Ribonuclease H Domain of the Hu an Immunodeficiency Virus Reverse Transcriptase Using 15N Relaxation Measurements

Robert Powers, G. Marius Clore, Angela Gronenborn, Stephen J. Stahl, Paul T. Wingfield

Research output: Contribution to journalArticlepeer-review

72 Scopus citations


The backbone dynamics of the uniformly 15N-labeled ribonuclease H (RNase H) domain of human immunodeficiency virus (HIV-1) reverse transcriptase have been investigated using two-dimensional inverse-detected heteronuclear 15N-1H NMR spectroscopy. 15N T1, T2, and nuclear Overhauser enhancement (NOE) data were obtained for 107 out of a total of 134 backbone amide groups. The overall rotational correlation time (TR) for the protein at 26 °C is 10.4 ns. The backbone N-H vectors for all the measurable residues exhibit very fast motions on a time scale of ≤20 ps. The 15N relaxation data for only 14 residues can be explained by this single internal motion alone. A further 39 residues display a second motion on a time scale ranging from 28.8 ps to 3.9 ns, while another 15 residues are characterized by an additional motion on the 170-ns to 2.25-ms time scale resulting in 15N T2exchange line broadening. There are 39 residues that exhibit both the additional 15N T2exchange line broadening and the slow (28.8 ps-3.9 ns) internal motion. Thus, the RNase H domain experiences extensive mobility throughout its structure as evidenced by the 93 residues which exhibit multiple modes of motion. Distinctly mobile regions of the protein are identified by large decreases in the overall order parameter (S2) and correspond to the C-terminal residues and the loop regions between β-strands β1and β2and between α-helix and β-strand β4. The high mobility of the C-terminus is of particular interest since one stretch of the sequence in this region of the protein constitutes part of the proposed substrate binding site. Thus, a highly flexible or partially folded binding pocket could explain the lack of enzymatic activity observed for this particular HIV-1 RNase H domain.

Original languageEnglish (US)
Pages (from-to)9150-9157
Number of pages8
Issue number38
StatePublished - Feb 1 1992
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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