Structural investigation of g-protein signaling in plants
Kaplan Türköz, Burcu (2009) Structural investigation of g-protein signaling in plants. [Thesis]
Heterotrimeric G proteins, composed of alpha, beta and gamma subunits, are a major group of signaling molecules in eukaryotic organisms. There is lack of direct biophysical and structural data for the plant heterotrimer unlike its mammalian counterparts. Heterotrimeric G protein subunits from Arabidopsis were cloned and purified. The alpha subunit, GPA1 was purified from Pichia, with a GTP binding ratio of 0.3 mole GTP/ mole protein. The recombinant beta (AGB1) and gamma (AGG1 and AGG2) subunits were isolated from E.coli and preliminary purification strategies were optimized. This is to our knowledge, the first study to report recombinant production of a plant beta subunit and in vitro dimerization of purified AGB1-AGG2 subunits. GPA1 was purified in two different biophysical states, as characterized by UVspectroscopy, dynamic light scattering, circular dichroism spectropolarimetry and mass spectrometry. The stable oligomeric form had higher GTP hydrolysis activity and a GDP binding ratio of 1.4 mole GDP/mole protein. Indirect biophysical evidence points to interaction of GPA1 with receptor mimetic compounds, membrane fractions of yeast cells and the recombinant AGB1-AGG2 dimer. This is to our knowledge, the first study showing the expression and purification of the plant alpha subunit from a eukaryotic expression system and its detailed biophysical characterization. Small angle solution X-ray scattering (SAXS) measurements verified the oligomeric nature of the protein, which was stabilized via detergent micelles. The detergent content was verified by proton nuclear magnetic resonance spectroscopy. SAXS patterns were consistent with dimeric protein complexing with micelles. Rigid body modeling was used for further modeling.
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