The objective of this proposal is to elucidate the structure and functions of a membrane domain composed of complexed poly-beta- hydroxybutyrate/calcium polyphosphate (PHB/DaPolyPi) associated with proteins. The complexes are widely distributed in the plasma membranes of prokaryotes, and in the mitochondria, microsomes and plasma membranes of eukaryotes. The isolated complexes conduct calcium currents when incorporated into phospholipid bilayers; in vivo, the complexes are postulated to function in Ca2+ transport, Ca2+ storage and DNA transport. Structural studies will address complexation between Ca2+, PHB and PolyPi, structural uniformity, the conformation of PHB, and the helical pitch and dimensions of the unit cell. Strategies for structural analysis will include 1H and 31P NMR, and FT-IR of solubilized complex; 13C-MAS NMR of complex formed in vitro in membrane vesicles; microscope-aided FT-IR, and X-ray powder diffraction patterns of solid complex. The coordination sphere of Ca2+ in the complex will be investigated through examination of cation selectivity, mobility and replacement employing 45Ca2+ and atomic absorption spectroscopy to analyze cation compositions. The role of the complex in Ca2+ storage and transport will be examined in E. coli cells and membrane vesicles. E. coli become genetically competent in response to a Ca2+ pulse which triggers synthesis of PHB and PolyPi. The polymers sequester Ca2+ to form polymer salt complexes within the membrane. The driving force(s) for PHB/CaPolyPi synthesis and Ca2+ transport will be analyzed using spectrofluorometry, atomic absorption spectroscopy, and radiolabelling methodologies with Ca2+ indicators, ionophores, Ca2+ antagonists, proton-conducting uncouplers, indicators of intracellular pH and membrane potential, and inhibitors of respiration and oxidative phosphorylation. Finally, the changes in intracellular Ca2+ and pH immediately following the Ca2+ pulse will be examined in individual cells using the fluorescent indicators, Indo-1 and SNARF, employing an interactive laser cytometer.
Showing the most recent 10 out of 16 publications
