Polarized In meso crystallization and lipid composition of recombinant human α4β2 nicotinic acetylcholine receptor detergent complexes

Abstract

Obtaining high-resolution 3D structures of membrane proteins through X-ray crystallography remains a longstanding bottleneck in the field of structural biology. This challenge has led to the optimization of purification methods to acquire high-yielding, pure proteins suitable for crystallization. In this work, we establish the expression of the recombinant version of the neuronal α4β2 nAChR through baculovirus transductions of mammalian cells, with the goal of producing purified milligram concentrations of the receptor, suitable for crystallization trials. Before purification, every solubilized sample of the receptor was evaluated through Fluorescence Size Exclusion Chromatography (FSEC) to characterize its expression and integrity. Using these purified fractions, we performed crystallization screenings of human α4β2 nAChR using a polarized in meso method. After reconstituting the detergent-solubilized α4β2-nAChR into the LCP matrix, the samples were incubated in a polarized lipid matrix using the RMP@LMx device developed in our laboratory. The results showed that under these conditions, the α4β2-nAChR-LFC16 complex gave a mobile fraction >0.8 as measured through Fluorescence Recovery After Photo-bleaching (FRAP) experiments, suggesting that its diffusion in the polarized lipid matrix is favorable for crystal nucleation.

During the polarized in meso crystallization screenings, we saw that voltages above 70 mV restrict crystal formation due to sample dehydration. Furthermore, a lipid analysis using UPLC-ESI MS/MS revealed a profile necessary for preserving protein integrity and promoting diffusion across the LCP. We harvested a single crystal and several “crystal-like” samples which were subjected to X-ray diffraction at beamline 23-ID-D at Argonne National Laboratory – Advanced Photon Source, resulting in reflections comparable to previous studies of the muscle-type nAChR from Torpedo californica. X-Ray diffraction of the single crystal gave distinct patterns consistent with a “fiber-type” diffraction composed of detergent-protein aggregates, as well as low-resolution reflections that might be of protein nature. These findings lay the groundwork for further optimization of protein membrane protein crystallization in polarized in meso phases.