Thermodynamics of stacking and of self-association of the dinucleoside monophosphate m2(6)A-U from proton NMR chemical shifts: differential concentration temperature profile method.

Chemical shifts of base and sugar protons of the modified ribodinucleoside monophosphate N6-dimethyladenylyl(3'-5')uridine (m2(6)A-U) were measured at 100, 360 and 400 MHz in aqueous solution. Seven different samples were used with concentrations ranging from 0.28 mM to 32.7 mM. The temperature was varied from -5 degrees C to 105 degrees C. An internal temperature calibration was used. The effects of intermolecular self-association and of intramolecular stacking on the chemical shifts were quantitatively separated by means of a new approach: differential concentration/temperature profiles (DCTP). Several computational models were tested and the analysis allowed deeper insight into the behaviour of m2(6)A-U at the molecular level. The simple two-state approach for both self-association and stacking already afforded a significant improvement over models in which the association is entirely neglected. A computer least-squares analysis of the chemical shift behaviour of each individual proton yielded thermodynamic parameters for self-association and stacking. However, the two-state model did not suffice to reproduce accurately all of the observations. A satisfactory fit required two additional assumptions: (a) the aromatic protons experience different association shifts in stacked and in unstacked molecules: (b) a temperature-dependent conformational equilibrium exists between sets of unstacked microstates. The stacked state is taken to represent a single conformational species. The implementation of this extended model in the least-squares optimization allowed the reproduction of over one thousand chemical shift observations within experimental error. Thermodynamic equilibrium parameters deduced for intramolecular stacking are: delta H degrees x = -28.8 kJ mol-1, delta S degrees x = -93 J mol-1 K-1. These numbers agree well with those obtained earlier by us from circular dichroism spectra. The equilibrium enthalpy and entropy values deduced for the association process are: delta H degrees A = -35 kJ mol-1 and delta S degrees A = -95 J mol-1 K-1.