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 finge r motif, and the BH10 NCp7 contains an additional Trp, at position 61 in th e C-terminal chain. The time-resolved intensity decay parameters of the zin c-saturated BH10 NCp7 were determined and compared to those of single-Trp-c ontaining derivatives. The fluorescence decay of BH10 NCp7 could be clearly represented as a linear combination (with respect to both lifetimes and fr actional intensities) of the individual emitting Trp residues. This suggest ed the absence of interactions between the two Trp residues, a feature that was confirmed by molecular modeling and fluorescence energy transfer studi es. In the presence of tRNA(Phe), taken as a RNA model, the same conclusion s hold true despite the large fluorescence decrease induced by the binding of tRNA(Phe). Indeed, the fluorescence of Trp(37) appears almost fully quen ched, in keeping with a stacking of this residue with the bases of tRNA(Phe ). Despite the multiple binding sites in tRNA(Phe), the large prevalence of ultrashort lifetimes, associated with the stacking of Trp(37), suggests th at this stacking constitutes a major feature in the binding process of NCp7 to nucleic acids. In contrast, Trp(61) only stacked to a small extent with tRNAPhe. Th, behavior of this residue in the tRNA(Phe)-NCp7 complexes appe ared to be rather heterogeneous, suggesting that it does not constitute a m ajor determinant in the binding process. Finally, our data suggested that t he binding of NCp7 proteins from the two HIV-1 strains to nonspecific nucle ic acid sequences was largely similar.