PHOSPHORESCENCE AND OPTICALLY DETECTED MAGNETIC-RESONANCE INVESTIGATION OF THE BINDING OF THE NUCLEOCAPSID PROTEIN OF THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 AND RELATED PEPTIDES TO RNA

The RNA and DNA complexes of nucleocapsid protein p7.Zn (NCp7.Zn) of the human immunodeficiency virus type 1 (HIV-1) are studied by phosphorescence and optically detected magnetic resonance (ODMR). The single tryptophan, Trp37, which is located on the C-terminal zinc finger domain is used as an intrinsic probe. Reductions in the triplet state zero-field splitting (zfs) D parameter of Trp37 upon complex formation with poly(I) and poly(U) are observed. These results, in conjunction with the phosphorescence red-shifts and triplet state lifetime reductions that are observed, suggest the presence of aromatic stacking interactions between NCp7.Zn and the bases of the RNA polymers. An alteration of the intersystem crossing pattern upon complex formation, in addition to the above mentioned spectroscopic shifts, also is consistent with previously observed tryptophans that undergo stacking interactions with DNA bases [Zang, L.-H., Maki, A. H., Murphy, J. B., and Chase, J. W. (1987) Biophys. J. 52, 867-872. Tsao, D. H. H., Casas-Finet, J. R., Maki, A. H., and Chase, J. W. (1989) Biophys. J. 55, 927-936]. These conclusions support those from a recent ODMR study [Lam, W.-C., Maki, A. H., Casas-Finet, J. R., Erickson, J. W., Sowder, R. C., II, and Henderson, L. E. (1993) FEBS Lett. 328, 45-48] of NCp7.Zn binding to 5-mercurated polyuridylic acid [poly(5-HgU)] in which stacking interactions between the RNA and NCp7.Zn are inferred from the observation of an external heavy atom effect induced on Trp37. The extent of the spectroscopic effects observed varies with different RNA complexes; the phosphorescence red-shifts, for instance, correlate with the affinities of NCp7.Zn for various RNA bases as measured by fluorescence quenching experiments [Casas-Finet, J. R., Sowder, R. C., II, Sakaguchi, K., Appella, E., Henderson, L. E., and Erickson, J. W. (1993) Biophys. J. 64, A178]. The complexes of an 18mer synthetic second zinc finger peptide of NCp7 with RNA polymers gave results similar to NCp7.Zn, indicating that tryptophan in either the wild type protein or in the synthetic peptide experience similar environments. However, spectroscopic effects of smaller magnitude are observed in the synthetic second zinc finger peptide complexes, relative to those in the NCp7.Zn complexes, suggesting that the two zinc fingers in NCp7.Zn may act in concert to bind RNA, A synthetic carboxymethylated second zinc finger peptide in which a zinc finger structure cannot be formed also is studied. The triplet state properties observed for the uncomplexed synthetic carboxymethylated second zinc finger peptide are similar to those of the noncarboxymethylated synthetic second zinc finger peptide, suggesting that the tryptophans in the two fingers have similar environments in the uncomplexed form. When either poly(I) or poly(U) is added to the synthetic carboxymethylated second zinc finger peptide, practically no spectroscopic effects are observed, indicating weak or no interaction between Trp37 and the RNAs under experimental conditions similar to those used for NCp7.Zn and the synthetic second zinc finger peptide binding.