Does tryptophan intercalate in DNA? A comparative study of peptide binding to alternating and non-alternating A.cntdot.T sequences

Abstract

The interactions of a tetrapeptide, lysyltryptophylglycllysine tert-butyl ester (KWGK), with synthetic double-stranded polynucleotides [poly(dA) .cntdot. poly(dT), poly[d(A-T)], poly(rA) .cntdot. poly(dT), and poly(rA) .cntdot. poly(rU0], Escherichia coli DNA, and single-stranded polynucleotides [poly(rA), poly(rU), poly(dA), and poly(dT)] were studied in a low-salt buffer by absorption and fluorescence spectroscopy. From fluorescence quenching data, we determined the two binding constants K1 and K2 of the two-step mechanism previously proposed for lysyltryptophyllysine binding to polynucleotides [Brun, F., Toulme, J. J., and Helene, C. (1975) Biochemistry 14, 558-563]. The first complex (PN)1 is purely due to electrostatic interactions between the lysyl residues and the phosphate groups. The second complex (PN)2 involves an additional stacking of the indole moiety of the tryptophyl residue with the bases (or base pairs) of the polynucleotide and is in equilibrium with (PN)1. K2 measures the ratio of the concentrations of stacked and unstacked complexes. The fluorescence decay of the tryptophyl residue in KWGK was not significant different in the presence and in the absence of double-stranded polynucleotides in agreement with the previous model, which assumes total quenching of tryptophan fluorescence in complex (PN)2 and identical fluorescence characteristics for free KWGK and complex (PN)1. The stacking of the tryptophyl residue with A-T base pairs in alternating poly[d(A-T)] was found to be 10 times more efficient than that with nonalternating poly(dA) .cntdot. poly(dT). Among A-T-containing double-stranded polynucleotides, poly(rA) .cntdot. poly(dT) was found to be the most favorable for tryptophan stacking. A similar behavior was previously demonstrated for several intercalating agents such as ethidium bromide, propidium iodide, and daunomycin. We demonstrated that quinacrine also behaved similarly; its binding decreased in the order poly(rA) .cntdot. poly(dT) > poly[d(A-T)] > > poly(dA) .cntdot. poly(dT). With single-stranded polynucleotides, tryptophan stacking decreased in the order poly(dT) > poly(rU) > poly(rA) > poly(dA). These results are discussed with respect to intercalation of the tryptophyl residue between A .cntdot. T base pairs and to the differences in conformation between alternating and nonalternating A .cntdot. T-containing polymers. The tryptophyl residue behaves as an intercalating agent with strong preference for alternating as compared to nonalternating A .cntdot. T base pairs in the deoxyribo polymers. Stacking of tryptophyl residues of DNA-binding proteins might be involved in base sequence discrimination.