Characterization of A. thaliana G-Protein gamma subunit (AGG2) and investigation of DTT effect on its oligomeric state
Aktürk, Anıl (2012) Characterization of A. thaliana G-Protein gamma subunit (AGG2) and investigation of DTT effect on its oligomeric state. [Thesis]
Official URL: http://192.168.1.20/record=b1477029
Heterotrimeric G-Proteins are vital effectors in signal transduction pathways in both animal and plants by transmitting the signal received by membrane bound receptors to downstream factors (Simon M. I., 1991). The protein complex consists of three subunits: α, β and γ. The alpha subunit has GTP hydrolysis activity and acts as a molecular switch for signal continuity or inhibition. Beta and gamma subunits act as a dimer for downstream signal transduction (Oldham, 2006). Odor, taste and phototransduction pathways are closely integrated to G-Protein transduction mechanism in mammalian organisms (Arshavsky V. Y., 2002). Plant cells utilize G-Protein signaling in germination, cell division, stress responses and morphological changes (Perfus-Barbeoch L., 2004). This study is the first report in the literature for biophysical characterization of the A. thaliana G-Protein γ subunit, AGG2, independent of beta subunit. The gene was expressed in E. coli TOP10 cells using pETM-41 and pMCSG-7 vectors. Expression profiles of the proteins and growth curves of TOP10 cells with or without protein overexpression were optimized. The proteins were purified with affinity, ion exchange and size exclusion chromatography and were characterized with Dynamic Light Scattering (DLS), Circular Dichroism (CD) and Small Angle X-Ray Scattering (SAXS) methods. Furthermore alignment of AGG2 to mammalian G-Protein, transducin, gamma subunit was carried out and this was utilized for homology modeling. The monomer model was self-docked and the structural features of the dimer were further investigated. Protein expression levels were such that the pETM-41 clone yielded insufficient protein for purification but pMCSG-7 clone (AGG2) and a previously prepared pQE80-L clone (AGG2*) could be utilized in biophysical characterization studies. Proteins AGG2 and AGG2* were obtained from the SEC column at different elution volumes indicating different properties for the same protein under the different conditions of SEC. This effect was traced back to presence/absence of DTT in elution buffers. Presence of DTT in the buffer resulted in a change in the size of the protein. DLS, SAXS, CD measurements as a function of DTT concentration (from 0 to 5 mM) showed that the protein changed its oligomeric state from tetramer to dimer as DTT concentration is increased and it has been possible to reduce this form to monomeric state. This effect was found to be independent of protein concentration. Homology modeling resulted in structures that were in agreement with those found from ab initio modeling based on SAXS data as well as that obtained from crystal structure of mammalian beta-gamma dimer. Theoretical SAXS curves obtained from the homology model of the dimer overlap well with the experimental SAXS curves. Significance of the dimer for function of the gamma subunit is discussed within the perspective of available literature on the plant G-protein heterotrimer. Modeling and biophysical characterization studies led to the conclusion that the dimeric form results from interaction of Cys108 of two monomers which are located in an intertwined ball like structure formed by C-termini loops of two monomers.
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