Investigation of structural properties of heterotrimeric G-Proteins y subunits in plants
Avşar, Bihter (2017) Investigation of structural properties of heterotrimeric G-Proteins y subunits in plants. [Thesis]
In plants, heterotrimeric G proteins are involved in the transmission of signals that activate several signaling pathways including those regulating seed germination and size, growth, differentiation, and responses to biotic and abiotic stress. Similar to their mammalian counterparts the canonical plant heterotrimeric G proteins consist of alpha (G), beta (G) and gamma (G) subunits. Upon activation G and G and G , as a heterotrimeric complex, interact with downstream effectors to activate signaling pathways. Although plant G proteins have been identified at the genomic level and several in vivo functional studies have been carried out, there is no data available on the biochemical and structural characterization of the plant G and G subunits. On the other hand, recently there is increased interest in the plant G subunits due to the discovery of several classes and also their direct involvement in plant stress responses. This thesis represents the first example in the literature of a comparative experimental investigation of biochemical characteristics and the structures for AGG1, the subunit from the model organism A.thaliana and RGG1 and RGG2, those from O.sativa independent of the presence of the respective G subunits. The AGG1, RGG1 and RGG2 genes were cloned into different plasmids for expression in Ecoli. The sufficient amount of protein was purified, and structural characterization studies were conducted. The oligomeric nature of the proteins from dimers, tetramers to the higher order or nonspecific aggregates were demonstrated in SDS- and native-PAGE and dynamic light scattering (DLS) results. DLS measurements indicated that with fresh samples the lowest oligomeric species that can be captured is the dimer with hydrodynamic radii of about 6:8 0:5 nm and 5:1 0:4 nm for RGG1 and RGG2, respectively. Circular dichroism (CD) experiments and secondary structure analyses were performed. Computational work was also carried out for determination of evolutionary relationships among the proteins and even to make predictions about their structures by homology modeling. Models for AGG1 and RGG1 proteins based on a structure derived for mammalian heterotrimeric subunit yielded helical structures with loops and disordered regions that were consistent with the CD results. Due to the lack of structures for homologous proteins for RGG2 ab initio modeling was undertaken. Results obtained from structural characterization are discussed from the broader perspective of a model for plant subunit structure-function relationships that was developed from our previous work.
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