Ligand-Induced Internal Molecular Dynamics of Nicotinic Acetylcholine Receptor Analysis by Diffracted X-Ray Tracking

Abstract

The nicotinic acetylcholine receptor (nAChR) is a pentameric ligand-gated ion channel in the central and the peripheral nervous system. The gating mechanism of nAChR is still unclear since the structure of nAChR in the presence of acetylcholine (ACh) has not been determined yet. Additionally, single molecular dynamics of nAChR in the presence of acetylcholine was not observed. In this study, diffracted X-ray tracking (DXT) was applied for nAChR system. DXT is a method to track the X-ray diffraction spot from the gold nanocrystal labeled on an individual single protein and enables us to observe intermolecular dynamics of the protein in real time and real space. Dynamic twisting motions upon gating of KcsA, a pH sensitive potassium channel, were successfully revealed by the DXT [Shimizu et al. (2008). Cell 132, 67-78]. At first step of new experiments, acetylcholine-binding protein (AChBP) was used as a model system of nAChR. AChBP is a structural and functional homologue of extracellular ligand-binding domain of nAChR. We investigated internal motions of AChBP by DXT in the absence and the presence of ACh, and found that ACh significantly activated the motion of AChBP. This result indicates that the ligand binding may initiate vigorous molecular fluctuations in AChBP, which was also confirmed by molecular elasticity measurement of AChBP with atomic force microscopy (AFM). In order to apply the DXT for the full length of nAChR, technological innovations will be incorporated into the DXT, such as efficient preparations of gold nanocrystals and speedup of the time resolution of DXT (5-10 μs). Additionally, we can control the orientation of adsorbed nAChR. The improvement of our technology and experimental results will be discussed.