Monitoring tat peptide binding to TAR RNA by solid-state 31P-19F REDOR NMR.
Complexes of the HIV transactivation response element (TAR) RNA with the viral regulatory protein tat are of special interest due in particular to the plasticity of the RNA at this binding site and to the potential for therapeutic targeting of the interaction. We performed REDOR solid-state NMR experiments on lyophilized samples of a 29 nt HIV-1 TAR construct to measure conformational changes in the tat-binding site concomitant with binding of a short peptide comprising the residues of the tat basic binding domain. Peptide binding was observed to produce a nearly 4 A decrease in the separation between phosphorothioate and 2'F labels incorporated at A27 in the upper helix and U23 in the bulge, respectively, consistent with distance changes observed in previous solution NMR studies, and with models showing significant rearrangement in position of bulge residue U23 in the bound-form RNA. In addition to providing long-range constraints on free TAR and the TAR-tat complex, these results suggest that in RNAs known to undergo large deformations upon ligand binding, 31P-19F REDOR measurements can also serve as an assay for complex formation in solid-state samples. To our knowledge, these experiments provide the first example of a solid-state NMR distance measurement in an RNA-peptide complex.
Olsen
Greg L
GL
Department of Chemistry, University of Washington Seattle, WA 98195-1700, USA.
Edwards
Thomas E
TE
Deka
Pritilekha
P
Varani
Gabriele
G
Sigurdsson
Snorri Th
ST
Drobny
Gary P
GP
D001665:Binding Sites; D005461:Fluorine; D015696:Gene Products, tat / Q000737:chemistry* / Q000378:metabolism; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010759:Phosphorus Isotopes; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D010455:Peptides; D010759:Phosphorus Isotopes; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; D005461:Fluorine