Eu3+ and Tb3+ ion luminescence is exploited to probe structural and functional aspects of proteins normally associated with the spectroscopically silent Ca2+ ion, as well as those which bind other dipositive ions. Eu3+ is excited to luminesce using a powerful visible wavelength Nd-YAG pumped pulsed tunable dye laser. Experiments may be carried out at submieromolar concentrations of protein. They yield information regarding metal ion binding constants, stoichiometry, total charge on liganding groups, numbers of coordinated water molecules, and distances between binding sites (from energy transfer experiments). Tb3+ ion luminescence sensitized via energy transfer from fluorescent amino acid residues (Tyr or Trp) is also used in distance measurements. Experiments involving collision-induced energy transfer between Eu3+ bound at a Ca2+ - binding site in a protein and small inorganic acceptor ions eg. [Co(NH3)6]3+ freely diffusing in solution are used to probe binding site accessibility and protein surface charge distribution. The use of chiral chelate complexes as acceptors will probe chiral recognition in the binding site region. In-depth studies of the important regulatory protein calmodulin and its interaction with drug molecules, peptide antagonists, and target enzyme analogs will be carried out in order to establish the solution structures of these complexes. An investigation of the protein phosphatase calcineurin with its calmodulin-like B subunit and catalytic transition metal ion-binding A subunit will be undertaken. Collaborative projects with other research groups involve glutamine synthetase and T7 phage DNA polymerase where we are exploring the effects of site directed mutations on the divalent metal ion binding sites. Ion selectivity in the channel-like structures of icosahedral viruses including the satellite tobacco necrosis virus will be studied using our laser techniques. The new area of 89Y nmr as a probe of Ca2+ binding sites is being explored by means of experiments on parvalbumen and calmodulin. Molecular mechanics/dynamics parameters for Ca2+ and the lanthanide ions binding to proteins are being developed via simulation of small molecule X-ray structures (with full three-dimensional periodicity) and solution thermodynamics and structure. These calculations will provide insight into metal ion selectivity and the changes which occur upon the introduction of a probe ion species.
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