Charles M. Grisham

Biophysical Chemistry; Magnetic Resonance Spectroscopy of Complex Biological Structures,There are currently two fundamental directions to our research. In one of these, biological membranes and complex biomolecules are being studied using nuclear magnetic resonance (NMR) and electron spin resonance (ESR) techniques. Current areas of interest include two ion transporting enzymes (kidney Na,K-ATPase and muscle Ca-ATPase), and two membrane-associated signalling enzymes (protein kinase C and phospholipase C. The ATPases use the free energy of hydrolysis of ATP to transport sodium, potassium or calcium across cell membranes against large concentration gradients. Protein kinase C is an intracellular mediator of hormonal and neurotransmitter stimuli and is also the receptor for phorbol ester tumor promoters. The geometry and active site structures of these complex systems are being examined by several methods. In one, we employ paramagnetic probes, such as Mn and Gd ions, Cr-nucleotide complexes and spin label analogues of enzyme substrates and inhibitors. Such probes perturb the nuclei in their vicinity and alter the nuclear relaxation rates. Quantitation of such effects can provide distances between the probes and nuclei on the enzyme surface. Another method, transferred nuclear Overhauser enhancement, permits additional studies of the conformation of substrates and activators at the active sites of these enzymes. One of the most interesting of these membrane-associated enzymes is the adenylyl cyclase toxin from Bordetella pertussis. Attack of host cells by this toxin results in transport of the catalytic domain of this toxin across the plasma membrane. The mechanism of this transport is not understood, but it appears to depend on a family of b-sheet helix domains in the C-terminal portion of the toxin. We are characterizing the structure and function of the b-sheet helices of this toxin by a variety of magnetic resonance techniques. We are also examining a series of metal complexes with organic bisphosphonates as potential therapeutic agents for osteoporosis and other bone diseases. Strength and integrity of bones depends upon a balance between bone formation by osteoblasts and bone resorption by osteoclasts. Osteoclasts depend for their activity on GTP-binding proteins Rho, Rab, and cdc42, which must be prenylated to be active. Prenyl groups are synthesized in the farnesyl pyrophosphate synthase (FPS) reaction. Bisphosphonates inhibit the FPS reaction and thus inactivate osteoclasts, which then undergo apoptosis, resulting in reduced bone resorption, lower bone turnover, and a positive bone balance. Stable complexes of bisphosphonates with Cr(III), Co(III), and Rh(III) are being examined as therapeutic alternatives to the metal-free bisphosphonates.