The interaction of HIV-1 Tat(32-72) with its target RNA: a fluorescence and nuclear magnetic resonance study.
We performed intrinsic peptide fluorescence experiments to characterize the interaction between variants of the HIV-1 Tat(32-72) peptide BP1 and TAR RNA. Kd values for wild-type BP1 and cysteine-modified BP1 were found to be in the range of 60 to 70 nM for both peptides, indicating that free sulfhydryl groups of the cysteines within the peptide are not required for high affinity TAR binding. Thus, the mutant peptide BP1 (C34S, C37W) (BP1SW) was used to further investigate peptide RNA interaction by fluorescence studies. Titration of BP1SW with TAR resulted in a dissociation constant (Kd = 9 nM) nearly an order of magnitude lower than that of the wild-type peptide. The change of the BP1SW 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 diffusion-limited initial formation of a tight, but unspecific peptide RNA complex, followed by a relatively slow structural rearrangement step (k approximately 60 s-1) in order to form the specific BP1SW-TAR complex. Comparison of heteronuclear two-dimensional NMR spectra of BP1SW and BP1SW bound to TAR shows that only resonances from amino acid residues of the core and basic sequence regions are shifted on TAR binding.
Metzger
A U
AU
Lehrstuhl für Biopolymere, Universität Bayreuth, Bayreuth, D-95440, Germany.
Bayer
P
P
Willbold
D
D
Hoffmann
S
S
Frank
R W
RW
Goody
R S
RS
Rösch
P
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D015696:Gene Products, tat / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D054322:tat Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015696:Gene Products, tat; D010446:Peptide Fragments; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; C109668:tat peptide (32-72), Human immunodeficiency virus 1