Screening for Torpedo Acetylcholine receptor crystals from electric organ: Structural studies on α7 neuronal nicotinic receptor
(Collaboration with Dr. Raymond Stevens, Scripps Clinics, San Diego, CA)
Membrane proteins and their complexes play crucial roles in many cellular and physiological processes. Functionally normal membrane proteins are vital to health, and specific defects are associated with many known disease states. Despite increases in the number of solved structures, the knowledge of membrane protein structures still lags far behind. The main goal of this project is to define how detergents affect the lipid composition and function of a model membrane protein and the ability to form crystals.
The objective of this project is to develop a comprehensive approach for the selection of detergents for membrane protein crystallization. The central hypothesis of the ongoing research is that detergents alter the native lipid composition during membrane protein solubilization and this could lead to irreversible damage to the lipid-spanning domains and eventually functional loss. During solubilization, a detergent may exclude a critical lipid specie(s) present in the normal cell membrane that is essential for protein stability and this could lead to a partial or permanent denaturation of the protein hydrophobic domains and thus disrupt its native conformation. The chemical nature (size, CMC, functional groups) of the detergent may be critical for lipid exclusion and the patterns observed in a model membrane protein could be used to develop correlations in other structure-related membrane proteins.
Our group has attempted to produce high-quality crystals of the Torpedo AChR for over a decade and the experience gained from a great deal of experimentation with this preparation has encouraged us to approach this problem from a unique perspective. Currently, we are studying the effects of a comprehensive list of detergents on: the Torpedo Acetylcholine receptor ion-channel activity, lipid composition, state of aggregation, potential contaminations and crystal formation. The information from these studies will provide a new perspective to define which detergents are suitable for AChR crystallization.
Over the past 2 years, we have also concentrated efforts to express a recombinant n-terminal α7 neuronal nAChR in bacteria, yeast and baculovirus. The rat neuronal α7 nAChR is a homomeric membrane protein of 5 subunits in a pentamer arrangement. Each subunit has an approximate molecular weight of 56 kDa with an extracellular domain of 208 residues, which contains the agonist binding site, 4 transmembrane segments (M1-M4) of approximately 20 residues and an M3-M4 intracellular domain of 152 residues. The primary objective of this project is to express and purify the rat α7 nAChR extracellular domain for crystallization trials. Guillermo Asmar, Ph.D., postdoctoral fellow working at Dr. Raymond Stevens’ laboratory (http://stevens.scripps.edu ) prepared a series of constructs that have been designed and cloned into E.coli, yeast and baculovirus (BV) vectors for expression and primary purification using immobilized metal affinity chromatography (IMAC). To test for activity and improve sample purity prior to crystallization trials, a bromoacetylcholine (BAC) affinity column derivatized from Bio-Rad Affi-Gel 10 will be used as the final purification step before coarse screen crystallization trials. We also perform functional studies using voltage clamp of Xenopus laevis oocytes expressing full-length α7 nAChR mutants.