Publication Date:
2012-11-04
Description:
HIV-1 reverse transcriptase (RT) contains a C-terminal ribonuclease H (RH) domain on its p66 subunit that can be expressed as a stable, although inactive protein. Recent studies of several RH enzymes demonstrate that substrate binding plays a major role in the creation of the active site. In the absence of substrate, the C-terminal helix E of the RT RNase H domain is dynamic, characterized by severe exchange broadening of its backbone amide resonances, so that the solution characterization of this region of the protein has been limited. Nuclear magnetic resonance studies of 13 C-labeled RH as a function of experimental conditions reveal that the 1 methyl resonance of Ile556, located in a short, random coil segment following helix E, experiences a large 13 C shift corresponding to a conformational change of Ile556 that results from packing of helix E against the central β-sheet. This shift provides a useful basis for monitoring the effects of various ligands on active site formation. Additionally, we report that the RNase H complexes formed with one or both divalent ions can be individually observed and characterized using diamagnetic Zn 2+ as a substitute for Mg 2+ . Ordering of helix E results specifically from the interaction with the lower affinity binding to the A divalent ion site.
Print ISSN:
0305-1048
Electronic ISSN:
1362-4962
Topics:
Biology
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