Heterotrimeric g-protein alpha (α) subunit from A. thaliana (AtGPA1) forms trimeric structures in solution
Çolak, Ersoy (2016) Heterotrimeric g-protein alpha (α) subunit from A. thaliana (AtGPA1) forms trimeric structures in solution. [Thesis]
The heterotrimeric guanine nucleotide-binding proteins (G-proteins) mediate transmission of signals from G protein coupled receptors (GPCRs) to effector systems including ion channels, enzymes and intracellular second messengers in yeast, mammals, and plants. The complex is comprised of alpha (Gα), beta (Gβ) and gamma (Gγ) subunits; Gα has GTP binding and hydrolysis activity, and Gβ and Gγ interact with downstream effectors as a dimeric complex. Although the structure and activation mechanism for the mammalian complex are well known, these are still not fully understood in plants. In our group subunits of the heterotrimeric complexes from Arabidopsis thaliana and Oryza sativa (rice) are heterologously expressed in yeast and bacteria respectively. The recombinant proteins are then purified for biochemical characterization and structural investigations. Our overall aim is to gain insight into the activation and signaling mechanisms of the G-protein complex in plants through structural studies on the individual subunits as well as the in vitro reconstituted complex. The work in this thesis is undertaken with the primary aim of optimizing the purification protocol of the A. thaliana Gα subunit, GPA1, expressed in yeast to obtain sufficient quantities of homogeneous recombinant protein for biochemical, biophysical, and structural analyses. A further goal is the recombinant production of a truncated version of the wild-type GPA1, which lacks the N-terminal 36 amino acids (GPA1t) in E. coli. GPA1t is commonly used in structural studies due to its stability. Results show that the optimized purification procedure has improved the yield to 5.5 mg GPA1 from 0.5 liters P. pastoris culture which represents a fivefold increase compared to earlier results in the group. Nucleotide (GTP, GDP, GTPγS) binding to the recombinant GPA1 is confirmed by absorbance spectroscopy and circular dichroism spectropolorimetry (CD). Results indicate that the secondary structure elements of GPA1 are more stable when it binds GTPγS as compared to GDP-bound form. Dynamic light scattering (DLS) results and Native-PAGE analyses combined with small angle X-ray scattering (SAXS) measurements reveal that GPA1 has a tendency to form trimers in solution. SAXS data also shows that GPA1-GDP has a globular structure with flexible regions extending from the protein. We cloned GPA1t gene in BL21 cells using the pQE80-L vector and a purification procedure was developed for the isolation of the recombinant protein. Despite the low protein yield, preliminary biochemical and biophysical characterizations were carried out on this protein. DLS measurements confirmed the smaller/more compact size of the GPA1t compared to GPA1. According to the CD analyses its thermal stability is higher than that of GPA1. In future studies conditions for the co-existence of the two species (monomer and trimer) and its physiological significance need to be investigated. Moreover, shape models for both species need to be developed to understand AtGPA1 interactions with other components in the heterotrimer.
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