Time-resolved fluorescence investigation of the human immunodeficiency virus type 1 nucleocapsid protein: influence of the binding of nucleic acids.
Depending on the HIV-1 isolate, MN or BH10, the nucleocapsid protein, NCp7, corresponds to a 55- or 71-amino acid length product, respectively. The MN NCp7 contains a single Trp residue at position 37 in the distal zinc finger motif, and the BH10 NCp7 contains an additional Trp, at position 61 in the C-terminal chain. The time-resolved intensity decay parameters of the zinc-saturated BH10 NCp7 were determined and compared to those of single-Trp-containing derivatives. The fluorescence decay of BH10 NCp7 could be clearly represented as a linear combination (with respect to both lifetimes and fractional intensities) of the individual emitting Trp residues. This suggested the absence of interactions between the two Trp residues, a feature that was confirmed by molecular modeling and fluorescence energy transfer studies. In the presence of tRNAPhe, taken as a RNA model, the same conclusions hold true despite the large fluorescence decrease induced by the binding of tRNAPhe. Indeed, the fluorescence of Trp37 appears almost fully quenched, in keeping with a stacking of this residue with the bases of tRNAPhe. Despite the multiple binding sites in tRNAPhe, the large prevalence of ultrashort lifetimes, associated with the stacking of Trp37, suggests that this stacking constitutes a major feature in the binding process of NCp7 to nucleic acids. In contrast, Trp61 only stacked to a small extent with tRNAPhe. The behavior of this residue in the tRNAPhe-NCp7 complexes appeared to be rather heterogeneous, suggesting that it does not constitute a major determinant in the binding process. Finally, our data suggested that the binding of NCp7 proteins from the two HIV-1 strains to nonspecific nucleic acid sequences was largely similar.
Laboratoire de Biophysique, URA 491 du CNRS, Faculté de Pharmacie, Université Louis Pasteur, B.P. 24, F-67401 Illkirch Cedex, France.
E
E
Bombarda
A
A
Ababou
C
C
Vuilleumier
D
D
Gérard
B P
BP
Roques
E
E
Piémont
Y
Y
Mély
D000595:Amino Acid Sequence; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002213:Capsid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009696:Nucleic Acids / Q000378:metabolism; D011485:Protein Binding; D012360:RNA, Transfer, Phe / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D009696:Nucleic Acids; D012360:RNA, Transfer, Phe; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus