Structural and thermodynamical analysis of molecular complexation by 1H NMR spectroscopy. Intercalation of ethidium bromide with the isomeric deoxytetranucleoside triphosphates 5′-d(GpCpGpC) and 5′-d(CpGpCpG) in aqueous solution
Abstract
An NMR analysis has been developed for determining the structural and thermodynamical parameters of molecular complexation in solution in situations where there is a multicomponent equilibrium. Using one-dimensional and two-dimensional 500 MHz 1H NMR spectroscopy, the method has been used to investigate complex formation between the phenantridinium dye, ethidium bromide (EB), and self-complementary deoxytetraribonucleoside triphosphates 5′-d(GpCpGpC) and 5′-d(CpGpCpG) in aqueous salt solution. Concentration dependences of proton chemical shifts of the molecules have been measured at constant temperature (T= 308 K) and the temperature dependence of chemical shifts (283–353 K) measured at constant concentration. Different schemes of complex formation between EB molecules and the tetranucleotides have been examined, successively taking into account various molecular associations in solution, viz 1 : 1, 1 : 2, 2 : 1 and 2 : 2 complexes. Equilibrium reaction constants and the limiting proton chemical shifts in the complexes have been determined. The relative contributions of the different types of complexes in the equilibrium mixture have been calculated with the 1 : 2 complex preferred for d(GCGC), which has one pyrimidine–purine binding sequence, whereas both the 1 : 2 and 2 : 2 complexes are significant for d(CGCG) which has two CG binding sequences. The induced limiting chemical shifts have been analysed in terms of the structures of the complexes. The results confirm that EB binds preferentially to the CG-sites of the tetranucleotide duplexes and that EB intercalates from the minor groove of the double-helix. These conclusions are supported qualitatively by 2D NOE measurements. The most favourable structures of the 1 : 2 and 2 : 2 dye–tetranucleotide complexes have been constructed using calculated values of induced chemical shifts of EB protons. The results are in good agreement with the structure of the EB–d(CpG)2 complex derived from X-ray crystallographic measurements. It is also found that EB binds to the single-stranded forms of the tetranucleotides in solution with formation of the 1 : 1 complex significant for d(GCGC) and both the 1 : 1 and 2 : 1 complexes forming for d(CGCG). The analysis shows that it is necessary to define all the reactions in a multi-component equilibrium if observed NMR parameters, which are weighted averages, are to be used to determine reliable structures of molecular complexes in solution.
The enthalpies and entropies of complex formation between EB and the tetranucleotides have been determined from the temperature dependence of the 500 MHz proton NMR chemical shifts. The contributions have been determined for the formation of different types of complexes (1 : 1, 2 : 1, 1 : 2 and 2 : 2) in solution. The analysis shows that the observed melting temperatures (Tm, which are also weighted averages) in the multi-component system are different from the values calculated for the 1 : 2 and 2 : 2 complexes. Comparison of the thermodynamical parameters has led to further understanding of the nature of the intermolecular interactions responsible for complex formation of EB with the different tetranucleotides.