Improved Purification and Crystal Formation of Native Muscle-Type nAChR Using mAbs

Abstract

The nicotinic acetylcholine receptors (nAChR) are part of the Cys-loop ligand-gated ion channel family and one of the most widely studied, but their molecular structure is still unknown. These receptors are important as the biological target for the development of therapies for a great number of diseases, including Myasthenia gravis, Parkinson's disease, and nicotine addiction. Our aim is to obtain high-quality protein crystals and determine the structure of the native nAChR via X-ray crystallography. We extracted muscle-type nAChRs from the tissue of Torpedo californica, using LFC-16 as a detergent, followed by several steps of purification. Four monoclonal antibodies (mAbs) with a high affinity against the receptor and the impurities were assayed via western blot, and BioLayer interferometry on the Octet platform for kinetic characterization. One of these mAbs yielded a KD <10−12 which suggests strong mAbs-nAChR interactions with no dissociation occurring. Furthermore, we developed a method of immunodepletion to remove impurities from the nAChR-detergent complex, increasing sample purity. Fluorescence recovery after photobleaching (FRAP) assays displayed a lower mobile fraction with mAbs incorporation compared to control, suggesting significant protein-protein interactions. Our next goal is to identify by western blot and protein fingerprinting which proteins are identified by the mAbs. These mAbs will be used to obtain high-quality nAChR crystals and also high-resolution X-ray data. This research was supported by the NIH NIGMS grants 1R01GM098343 (JALD); COBRE NIEF 1P20GM103642 (J.R and JALD) and Research Initiative for Scientific Enhancement (RISE) program.