THE INTERACTION OF HIV-1 TAT(32-72) WITH ITS TARGET RNA - A FLUORESCENCE AND NUCLEAR-MAGNETIC-RESONANCE STUDY

Citation
Au. Metzger et al., THE INTERACTION OF HIV-1 TAT(32-72) WITH ITS TARGET RNA - A FLUORESCENCE AND NUCLEAR-MAGNETIC-RESONANCE STUDY, Biochemical and biophysical research communications, 241(1), 1997, pp. 31-36
Citations number
31
ISSN journal
0006291X
Volume
241
Issue
1
Year of publication
1997
Pages
31 - 36
Database
ISI
SICI code
0006-291X(1997)241:1<31:TIOHTW>2.0.ZU;2-W
Abstract
We performed intrinsic peptide fluorescence experiments to characteriz e the interaction between variants of the HIV-1 Tat(32-72) peptide BP1 and TAR RNA. K-d values for wild-type BP1 and cysteine-modified BP1 w ere found to be in the range of 60 to 70 nM for both peptides, indicat ing that free sulfhydryl groups of the cysteines within the peptide ar e not required for high affinity TAR binding. Thus, the mutant peptide BP1 (C34S, C37W) (BP1(sw)) was used to further investigate peptide RN A interaction by fluorescence studies. Titration of BP1(sw) with TAR r esulted in a dissociation constant (K-d = 9 nM) nearly an order of mag nitude lower than that of the wild-type peptide. The change of the BP1 (sw) fluorescence intensity on TAR binding was used to investigate the kinetics of this interaction by stopped-flow experiments. The results can be explained in terms of a two-step binding model, with a rapid d iffusion-limited initial formation of a tight, but unspecific peptide RNA complex, followed by a relatively slow structural rearrangement st ep (k approximate to 60 s(-1)) in order to form the specific BP1(sw)-T AR complex. Comparison of heteronuclear two-dimensional NMR spectra of BP1(sw) and BP1(sw) bound to TAR shows that only resonances from amin o acid residues of the core and basic sequence regions are shifted on TAR binding. (C) 1997 Academic Press.