| P2X receptors are eukaryotic ligand-gated ion channels expressed throughout the nervous system. They are opened by extracellular ATP or its structural analogs to transmit signals for proliferation, differentiation, cell-death, and nociception. Despite a number of studies by electrophysiology, biochemistry, and cell biology, the molecular mechanism of ligand-gating and channel activities are unknown due to a lack of structural information. My thesis project was dedicated to screening various P2X constructs, optimizing expression/purification systems, and characterizing purified protein (precrystallization screening) with the goal of obtaining the three-dimensional (3D) structure of a P2X receptor. As it is extremely time and resource consuming to purify milligram quantities of a eukaryotic membrane protein, first I established an efficient precrystallization screening method in which the expression level, degree of monodispersity, and approximate molecular mass of the target protein are determined with nanogram quantities of unpurified protein as a GFP fusion.; Screening of expression constructs suggested that a C-terminal GFP fusion of P2X4 (C-P2X4) is promising for crystallization trials. A series of expression, purification, and characterization experiments have shown that C-P2X4 is functional, monodisperse, and stable in different detergents. However, removal of C-terminal GFP was problematic as thrombin digestion could not be completed. Consequently, P2X4 was purified without GFP fused to the C-terminus and characterized by biochemical experiments as well as electron microscopy. Interestingly, P2X4 forms a cluster when it is purified in n-dodecyl beta-D-maltoside (C 12M) but not in digitonin. Although crystallization trials in the presence of digitonin failed, probably because it is chemically unstable and crystallizes by itself in various conditions, supplementing cholesterol derivatives to detergents may be useful for crystallization trials of P2X 4 receptor. Finally, as an alternative approach for obtaining a 3D structure, single particle reconstruction of negatively stained P2X4 was performed. |
