PROGRESS TOWARDS CRYSTALLIZING A GABAA RECEPTOR

Purpose: Currently we lack a 3-dimensional template GABA interacting with the GABA-rho receptor. We are working towards determining the three-dimensional structure of the GABAA-rho receptor. This will assist in the development of novel therapeutics. Methods: GABA-rho1 was subcloned into a baculovirus expression vector using blunt end PCR and transfected into SF9 insect cells. SF9 cells were harvested, and protein was isolated using metal affinity and size exclusion chromatography. A phosphate buffered saline (PBS) based running buffer was used to stabilize the purified protein. Protein crystallization trials were performed using the lipidic cubic phase technique and observed weekly for crystal formation using a stereomicroscope. Results: GABAA-rho was isolated using metal affinity chromatography. Our size exclusion chromatography studies reveal that using a PBS-based running buffer stabilizes the pentameric arrangement of purified GABAA-rho receptor. Subsequent lipidic cubic phase crystallization trials revealed three conditions what yielded birefringent protein crystals. Conclusions: Previously, we determined the optimum incubation time to yield maximum amount of protein. However, the isolate protein was not stable in the subsequent purification steps. Here, we have identified optimal buffer and detergent conditions to isolate and solubilize the protein. We have also identified crystal conditions that yield crystals. Future experiments will focus on generating additional protein crystals in the presence and absence of ligands, such as GABA and GABAA-rho receptor selective antagonists.