Recombinant Expression and Characterization of A.thaliana Heterotrimeric G protein alpha subunit; GPA1
Kaplan Türköz, Burcu and Dinler-Doğanay, Gizem and Sayers, Zehra (2007) Recombinant Expression and Characterization of A.thaliana Heterotrimeric G protein alpha subunit; GPA1. In: Gordon Rsearch Conferences, Biddeford, ME, USA (Accepted/In Press)
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Current research on plant heterotrimeric G proteins is mainly focused on mutant / overexpression studies performed in model organisms, i.e. A. thaliana and O. sativa. These studies provide information about the pathways involving these proteins; however, the mechanism of G protein activation via signaling molecules remains unknown. Similarly, information on biochemical and biophysical characteristics of the plant heterotrimeric G-proteins is lacking, whereas for the mammalian counterparts, such studies have contributed unique insights into understanding their functional roles. Following this, A.thaliana heterotrimeric G protein subunit (GPA1) is cloned into P.pastoris expression system and the recombinant protein is purified. Initial analyses reveal that GPA1 is membrane bound and N-terminally blocked indicating the presence of lipid modifications. The purified protein is GDP bound and can bind to GTP. Circular dichroism polarimetry analyses show that the dominant structure is helical and activation by receptor mimetics leads to a decrease in helical content; similar to mammalian counterparts. It appears that, contrary to mammalian G-alphas, the intrinsic fluorescence of GPA1 decreases in the presence of GTP and increases in the presence of GDP. Full characterization of GAPA1, including its nucleotide binding kinetics and dynamics, will help to establish a better understanding of the functional mechanisms of heterotrimerc G-protein activation in plants and will shed light on the level of similarity of plant and mammalian hetetrotrimeric G protein signaling pathways.
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