Plant heterotrimeric G proteins are a major group of signaling molecules involved in regulation of critical processes including stress adaptation, seed size, grain quality and immune responses. Despite an abundance of in situ functional studies; purification of the individual subunits of the plant heterotrimer for biophysical and structural characterization and for studies on their interactions are lacking. In this study cloning of the genes encoding the β subunit AGB1 of A. thaliana and its γ-subunits AGG1 and AGG2 using different E. coli expression vectors and screening of expression in several strains are reported. AGB1 could be expressed albeit at very low levels and in all cases it was accompanied by overexpression of E. coli chaperone proteins. AGG1 could only be detected in inclusion body fractions, whereas AGG2 was obtained in soluble fractions and was purified. Purified AGB1 and AGG2 subunits were shown to dimerize in vitro. Further characterization of AGG2 by small angle X-ray scattering measurements and by dynamic light scattering revealed that AGG2 formed homodimers with extended shape in solution. These results are also consistent with those from circular dichroism spectroscopy which yielded 39.4% helical and 50% random coil content for AGG2. This is the first study showing heterologous expression of a plant heterotrimeric G protein β subunit individually and presenting its interaction with a plant γ-subunit in vitro. Results also show that the AGG2 subunit has a disordered structure, which would account for its role in diverse interactions for establishing selectivity in signal propagation.
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We would like to thank Dr. Dimitri Svergun and BIOSAXS group members at EMBL/DESY for their help in data collection and analysis and Dr. M. H. J. Koch for helpful discussions. This work was supported by the Sabanci University Internal Research Fund IACF08-00514 and by the bilateral program TUBITAK-Julich TBAG-U-155.
This work was supported by the Sabanci University Internal Research Fund IACF08-00514 and by the bilateral program TUBITAK-Julich TBAG-U-155
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Kaplan-Türköz, B., Aktürk, A. & Sayers, Z. Oligomerization of A. thaliana Heterotrimeric G Protein Subunits AGB1 and AGG2 In Vitro. Protein J 39, 563–573 (2020). https://doi.org/10.1007/s10930-020-09914-z
- Heterotrimeric G proteins
- Bacterial recombinant expression
- Biophysical characterization