H-1-NMR STUDIES ON ASSOCIATION OF MESSENGER-RNA CAP-ANALOGS WITH TRYPTOPHAN-CONTAINING PEPTIDES

H-1-NMR spectroscopy was applied to a study of the mode of interaction , in aqueous medium in the pH range 5.2-8.5 and at low and high temper atures, between several mono- and dinucleotide analogues of the mRNA c ap m(7)GpppG and a selected tripeptide Trp-Leu-Glu, and a tetrapeptide Trp-Glu-Asp-Glu, the sequence of which corresponds to one of the susp ected binding sites in the mRNA cap-binding protein (CBP). A program, GEOSHIFT, was developed, based on ring-current anisotropy theory, for analysis of experimentally observed changes in chemical shifts accompa nying interactions between aromatic heterocyclic rings. This permitted quantitative evaluation of stacking interactions between the m(7)G ca p and the tryptophan indole ring, and the relative orientations of the planes of the two rings, spaced about 3.2 Angstrom apart. The structu res of the stacked complexes were determined. In particular, stacking between m(3)(2,2,7)G (which has no free amino group for hydrogen bondi ng) and the indole ring is weaker and quite different from that betwee n m(7)G and m(2)(2,7)G and indole. With the dinucleotide cap-analogues , only the m(7)G component stacks with the indole ring, without disrup tion of intramolecular stacking, In contrast to numerous earlier repor ts, the calculated stacking interactions are quantitatively in accord with the values derived from fluorescence measurements. It also has be en shown that the positively charged (cationic) form of m(7)G stacks m uch more efficiently with the indole ring than the zwitterionic form r esulting from dissociation of the guanine ring NIH (pK(a) approximate to 7.3).