Insights into Crystallization of Neuronal Nicotinic α4β2 Receptor in Polarized Lipid Matrices

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 study, we performed crystallization screenings of purified 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-LFC 16 complex gave a mobile fraction >0.8, suggesting that its diffusion in the polarized lipid matrix is favorable for crystal nucleation. Voltages above 70 mV restricted 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 subjected it to X-ray diffraction, resulting in reflections comparable to previous studies of the muscle-type nAChR from Torpedo californica. X-ray diffraction of a single crystal gave distinct low-resolution diffractions of protein nature. These findings lay the groundwork for further optimization of membrane protein crystallization in polarized in meso phases.