Molecular-reorientational dynamics investigation of biomolecules in solution by NMR, X-ray and CSI-MS.

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I. An extensive analysis of hydrogen bonding of steroid compounds in diluted solution is preformed by PFG NMR, CSI-MS and in the solid state by X-ray crystallographic analysis. Although X-ray analysis in the crystalline state and CSI-MS measurement in solution suggested that the observed diffusion coefficient D_<obs> of the steroid compounds may vary in accordance with the number of hydrogen bonds, the actual observed D_<obs> value determined from the diffusion studies diminished constantly without correlation on the decreasing numbers of hydrogen bonds. Formation of a chain structure (cluster) based on intermolecular hydrogen bonding of the steroid compounds is unambiguously confirmed.II. To confirm this unprecedented phenomenon in solution, we performed diffusion study by PFG NMR. The D values of the complexes BG + nBA (n=1〜4) including BG and BA itself, obtained by PFG NMR measurements. The observed D value decreases in accordance with the increasing number of attached BA (n). This result clearly supports the ordered formation of the complexes in solution as observed by CSI-MS.III. It was found that ^<19>F{^1H} NOE and 2D ^1H-<-19>F HOESY experiments detected intermolecular NOEs between halothane ^<19>F and HSA protons. Measurements of diffusion coefficients for halothane were also carried out by ^1H and ^<19>F NMR, indicating the interaction of halothane with HSA. The present results indicate that these techniques are very suitable for identification of a fluorine containing ligand binding with a protein receptor in the drug discovery process.IV. The difference diffusion-based NMR technique can directly detect the ligand signals from the complex using a simple pulse sequence without the need for isotope labeling. The present results indicate that these techniques are very suitable for identifying a ligand binding with a protein receptor in the drug discovery process.