RUI: In Vivo and in Vitro Analysis of Modified Chloroplast Rieske Iron-Sulfur Proteins of Chlamydomonas

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The Rieske iron-sulfur protein of the chloroplast cytochrome b6 f complex has essential roles in energy transduction, redox-sensing, and in biochemical signaling to regulate photosynthesis. A unique movement of the iron-sulfur cluster domain is necessary for catalytic activity and probably for signaling. The research will investigate the role of protein structural features in signal transduction, catalytic activity, and assembly. Modified Rieske proteins will be studied in the alga Chlamydomonas, in isolated complexes, and as overproduced proteins in vitro. Specific objectives are: 1) Mutational investigations in vivo and in vitro of the role of the Rieske protein N-terminus and inflexible hinge domains in signal transduction and catalysis. Included are cysteine insertions to attach fluorescent probes to isolated complexes for assessment of possibleRieske N-terminal movement in signaling. 2) Mutational studies in vivo of an N-terminal Lys-Arg motif and its possible role in .pH-dependent, Rieske protein translocation across the thylakoid membrane. 3) Analysis of overproduced, modified Rieske proteins (such as those lacking the disulfide bridge) by spectroscopic methods to characterize structure/function relationships. Proteins of particular interest may be purified for structure determination. 4) In vitro reconstitution of overproduced Rieske proteins with Rieske-depleted cytochrome b6 f complex. Success would allow in vitro studies of Rieske protein interactions with neighboring subunits and possible LHCII kinase activation in signaling. The research is multifaceted, relies on stimulating collaborations, and will provide important new insights into fundamental mechanisms of photosynthesis and membrane proteins. It will contribute to complementary research and teaching activities at UW-Oshkosh and will provide excellent training opportunities for MS and undergraduate students.