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
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.