Modeling of Biomolecular Systems Interactions, Dynamics, and Allostery: Bridging Experiments and Computations - September 10-14, 2014, Istanbul, Turkey

Modeling of Biomolecular Systems Interactions, Dynamics, and Allostery Poster Session II

79-POS Board 32 Structural Features of the Gamma Subunit (AGG2) and Specificity of A. Thaliana Heterotrimeric G-protein Interactions Zehra Sayers , Sandra Quarantini, Hazal B. Kose, Burak Gur. Sabanci University, Istanbul, Turkey. Heterotrimeric G-proteins are involved in several essential signaling pathways in plants, including those controlling seed germination, growth and defense against pathogens. Despite detailed functional and structural information on mammalian G-proteins, structure-function characterization of the plant proteins is lacking. Moreover, although there are basic similarities in the signaling mechanisms, regulation of activity in the mammalian and plant G-proteins show important differences. We cloned and expressed A. thaliana subunits alpha, beta and gamma in E. coli for structural studies and here results on the gamma subunit AGG2 will be presented. Purified recombinant AGG2 shows a propensity to oligomerize in solution and as determined by SDS- and native-PAGE, size exclusion chromatography and dynamic light scattering a stable dimer is obtained only under fully reducing conditions. Small angle X-ray solution scattering (SAXS) measurements reveal that AGG2 dimer has an elongated flexible structure which can be modeled with the ensemble optimization method (EOM) used for intrinsically disordered proteins. Results indicate that AGG2 structure can be described as a population of inter- converting flexible conformers which all contain a rigid α-helix domain involved in the inter- molecular coiled coil interactions. Estimation of the secondary structure content from circular dichroism (CD) measurements agrees well with that predicted by homology modeling and SAXS models. Results on thermal stability of the protein monitored by CD measurements are also in agreement with the flexible structure predicted by EOM models. Structural models together with sequence analyses show the centrality of the AGG2 N-terminal region in establishing the coiled- coil interaction, crucial for binding to its natural partner AGB1. Our results also suggest that the disordered flexible regions may be part of the mechanism for conferring functional selectivity to the A. thaliana beta-gamma dimer at different location in the plant.

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