Expression And Purification Of GABA_B Receptor And Structural And Functional Studies Of The MFS Transporter YajR

y-aminobutyric acid (GABA) is a predominant inhibitory neurotransmitter in the central nervous system. Metabotropic GABAB receptors belong to Class C G protein coupled receptors(GPCRs), which play an important role in regulating synaptic transmission and membrane excitability in brain. Malfunction of GABAB receptors can lead to various neurological disorders, including anxiety, epilepsy, depression, spastic, drug addiction and cognitive disorder.GABAB receptors function as a heterodimer assembled by the subunit GBR1and GBR2. Both subunits have a large extracellular Venus flytrap (VFT) module, a seven transmembrane domain and a short intracellular carboxy terminus forming coiled-coil responsible for linking the two subunits. All known ligands bind to the cleft of GBR1VFT but not GBR2VFT, while GBR2TMs domain interacts with Gi/o protein. Detailed structural basis of ligand selectivity, allosteric modulation, dimerization and masking of the endoplasmic reticulum (ER) retention sequence at the GBR1C-terninus remains elucidated. In the Part I of this thesis, we expressed and purified full-length, VFT, TMs, C-terminus and VFT-TMs of the GABAB receptor using E. coli system and insect system, screened crystallization conditions and obtained initial crystals. Firstly, the VFT of GBR1and GBR2from Rat and C. elegans was expressed in the form of inclusion body using E.coli system, and after refolding, the soluble part was used to crystal screening. We expressed and purified the C-terminal of both GBR1and GBR2using E.coli system, confirmed the interaction between the coiled-coil region of GBR1and of GBR2by GST pull-down assay and size exclusion chromatography(SEC), and screened extensive conditions for crystallization. We expressed and purified full-length and variant truncations of GABAB receptor using SF-9insect system, and improved yield and homogeneity by fusing T4lysozyme (T4L) or cytochrome b562ril (Bril), then screened thousands of conditions to crystallize GABAB receptor and obtained initial crystals. Our purification work provides a reference for the further GABAB receptor purification, and our initial GBR TMs crystal has the potential to improve its X-ray diffraction ability.Transport proteins allow uptake of essential nutrients and ions, excretion of end products of metabolism and toxic substances, and communications between cells and the environment. Moreover, they provide essential constituents of energy-generating and energy-consuming systems. The major facilitator superfamily (MFS) is the largest family of secondary active transporters and is present in all life kingdoms. The first MFS protein three dimension (3D) structure, lactose permease (LacY) was reported in2003. From then on more than ten crystal structures of MFS proteins has been reported. Despite those structures solved in different conformations support the "Rocker-Switch" hypothesis of MFS conformation change when transporting substrates, detailed structure basis of the substrate transport and energy-coupling mechanisms remain to be elucidated. To address questions about the general mechanisms of MFS proteins, in Part II of this thesis, we investigate the crystal structure of MFS transporter YajR from E. coli.YajR is a putative proton-driven MFS transporter found in many Gram-negative bacteria. We expressed recombinant YajR in E. coli, extracted the protein from membrane using detergents (DDM, etc), purified with metal affinity chromatography and size exclusion chromatography (SEC). Protein sample of high purity and homogeneity was crystallized with eight different detergents using the hanging-drop vapor diffusion method. After extensive screening, YajR crystals diffracted X-ray well enough to collect data at synchrotron X-ray source. Initial phases were calculated with two-wavelength anomalous dispersion data sets, and the final crystal structure was refined at3.15A resolution. Besides the canonical12transmembrane helices core which are similar to previously reported MFS transporter structures, YajR contains a small domain located C-terminally to the TM core and possessing a ferredoxin-like fold(YAM). While a ferredoxin-like fold usually has a βαββαβ secondary structural pattern, the last β-strand is missing in the small domain at the C-terminal of YajR. Within the TM core, the N-domain formed by TMs1-6is related to the C-domain formed by TMs7-12through a pseudo two-fold symmetry (with the axis perpendicular to the membrane plane). Both N-domain and C-domain possess a internal pseudo two-fold symmetry (with the axis parallel to the membrane plane) connecting TMs1-3to4-6and TMs7-9to10-12, respectively. The cytoplasm ends of the N-domain and C-domain contacts closely while the periplasm ends are apart from each other. So our structure showed an outward facing conformation. The crystal structure enabled us to observe the most conserved motif (motif A) in MFS family in function. This observation prompts us to propose a general mechanism of conformational changes of MFS proteins.