The overreaching of this supplemental application is to achieve a high resolution structure of the Torpedo nAChR and its complexes with various nicotinic ligands and also develop the RMP@LMx methodology that could be used to crystallize and achieve high resolution structures of other biologically relevant membrane proteins. A high resolution structure of the nAChR and of its complexes with various nicotinic ligands is of crucial importance for the design of novel agents that target defined nervous-system pathologies such as Alzheimer?s disease, schizophrenia, depression, attention deficit hyperactivity disorder and tobacco addiction. During the past 4 years we have used our multi-attribute analytical platform to characterize about 50 nAChR- detergent complexes (nAChR-DCs). Our recent approach has examined five different attributes of the nAChR- DCs: (1) stability of in lipidic cubic phase (LCP)-FRAP experiments, (2) aggregation using electron microscopy (EM), (3) purity using SDS gels and Microfluidic Capillary Gel Electrophoresis (MCGE), (4) functionality by recording macroscopic currents in Xenopus oocytes and (5) a comprehensive lipidomic analysis. Due to hurricane?s Irma and Maria our main laboratories at the Julio Garcia Diaz (JGD) building at the UPR-RP campus were flooded, and due to damages to the JGD Building?s generator a significant amount of materials and equipment were lost. This laboratory has remained closed since Maria slammed P.R., on September 19- 20, 2017, due to the significant infrastructure damage caused to the roof of this JGD building. One week after Maria?s destructive path, my research group was relocated to the Molecular Science Research Center (MSRC) for continuing the production of nAChR crystals in accordance with the original research plan. We also lost a large amount of nAChR crystals (>150) a few days before our last trip to Argon Photon Source (APS), on December 16, 2017 due to electrical damage to our crystal incubator. The entire electrical grid in P.R. was devastated by hurricane Maria and during recovery of the grid the recurrent power failures have caused significant damage to many instruments in our laboratory. The present supplemental application focuses on the optimization of the nAChR-LFC16 complex according to the original plan of the application. We propose two Specific Aims: (1) to asses and optimize the quality of the nAChR-LFC16 crystals by fine tuning crystallization conditions and monitoring crystal composition, and (2) to perform high-throughput crystallographic screening of the optimized nAChR-DCs using vapor diffusion, LCP and the new RMP@LMx device. This project is highly innovative in its use of the RMP@LMx device, conceived I our laboratory (www.nachrs.org) to crystalize the nAChR in its closest physiological environment. The funds requested in this supplemental application will allow us to complete the optimization of the nAChR- LFC16 complex, publish 3 new manuscripts, continuing the process of competitively renewing the grant, but most important it will allow us to recover our path towards the nAChR high-resolution structure.
The proposed research is relevant to public health because a high-resolution structure of the nAChR is of crucial importance for the design of novel agents that target defined nervous system pathologies such as Alzheimer?s disease, schizophrenia, depression, attention deficit hyperactivity disorder, and tobacco addiction. Thus, the proposed research is relevant to NIH?s mission in that it will increase current knowledge about an important ligand-gated ion channel that could help extend healthy life and reduce the burdens of illness.
