THERMODYNAMIC AND STRUCTURAL-PROPERTIES OF R(ACC) AS REVEALED BY ULTRAVIOLET ELECTRONIC ABSORPTION, CIRCULAR-DICHROISM, H-1-NMR SPECTROSCOPY AND MONTE-CARLO SIMULATIONS

UV absorption, circular dichroism (CD) and H-1 NMR, associated with Mo nte Carlo (MC) molecular structure simulations have been applied to th e study of the trinucleoside diphosphate: r(ACC). The MC study which h as been conducted as a function of temperature, is based on random var iations of the nucleotide conformational angles, i.e. phosphodiester c hain torsional angles and sugar pucker pseudorotational angles. All of the chemical bond lengths and valence angles remained fixed during th e structural simulation, except those of the sugar pucker. Six differe nt initial structures have been selected in order to explore the molec ular conformational space as completely as possible. This simulation p rocedure led to distinct families of equilibrium conformations at 283, 298 and 318 K. The thermodynamical parameters such as variations in e ntropy, enthalpy and also melting temperature (Delta S-X(0), Delta H-X (0) and T-m) of the stacking (X) equilibrium were obtained from UV abs orption and circular dichroism (CD) spectra recorded over a 80K temper ature range. Chemical shifts (delta), vicinal coupling constants ((3)J (k,l)), and cross-relaxation rates (sigma(k,l)) of trimers were measur ed at 400.13 MHz over a range of concentrations (2-13mM) and temperatu res (283-333K). Least-squares fitting of the experimental chemical shi fts to simple models of association (A) and stacking equilibria allowe d separation of the variations in the delta values (Delta delta(X) and Delta delta(A)) due to either phenomenon. The three NMR data sets (De lta delta(X), (3)J(k,l)) and sigma(k,l) were then evaluated for the mi nima conformers obtained with the MC simulations. Theoretical values o f Delta delta(X) were estimated using the results of an ab initio stud y while the coupling constant data were simulated with Karplus-type eq uations. Finally, the relaxation data were simulated from the distance matrices using treatment for cases of both slow conformational exchan ge accompanied by rapid small-amplitude fluctuations about the minima structures. A consistent picture of the large amplitude deformations ( torsional angle variation) of these trimers has emerged from the prese nt study. Optimized conformational blends at 283, 296 and 318K were ob tained by least-squares fitting of the experimental data to the theore tical ones, while considering the populations as adjustable parameters . As it would be expected, the right-handed helical conformation (A-RN A type) is found to be the major stacked species, in the temperature r ange of 283 to 318K. Limited evidence for bulged structures has been o btained, whereas novel reverse-stacked and half-stacked conformers als o presented theoretical data compatible with the NMR observables of aq ueous r(ACC).