Abstract
We observed the dynamic three-dimensional (3D) single molecule behaviour of acetylcholine-binding protein (AChBP) and nicotinic acetylcholine receptor (nAChR) using a single molecule tracking technique, diffracted X-ray tracking (DXT) with atomic scale and 100 μs time resolution. We found that the combined tilting and twisting motions of the proteins were enhanced upon acetylcholine (ACh) binding. We present the internal motion maps of AChBP and nAChR in the presence of either ACh or α-bungarotoxin (αBtx), with views from two rotational axes. Our findings indicate that specific motion patterns represented as biaxial angular motion maps are associated with channel function in real time and on an atomic scale.
Highlights
The nanocrystal was immobilised on the C-terminus of acetylcholine-binding protein (AChBP) via a Met-tag (Figure 1a) and on the F(ab’)[2] fragment of the monoclonal antibody 35, which recognises the extracellular side of the nicotinic acetylcholine receptor (nAChR) a-subunit[18] (Figure 1b)
We immobilised an internal motion tracer, a gold nanocrystal, on the ligand-binding domain of nAChR and the C terminal of AChBP and found that the combined tilting and twisting motions of the proteins were enhanced by ACh binding
We determined that ACh induced tilting and twisting motions for both AChBP and nAChR, whereas aBtx restricted these motions for both proteins
Summary
Real Time Ligand-Induced Motion Mappings of AChBP and nAChR Using X-ray Single Molecule Tracking. I n addition to the static crystallographic information regarding a protein’s structure, dynamic information regarding a protein’s conformational changes would be helpful in elucidating the molecular mechanisms that regulate protein functions, such as ion channel gating and ligand-induced receptor activation Such dynamic information can be obtained using recently developed single molecule techniques[1] and can be predicted using molecular dynamics simulations[2]. To improve the monitoring precision and signal-to-noise ratio under physiological conditions, we have proposed a single molecule technique that utilises short wavelength probes, such as Xrays[3,4] and electrons[5], to monitor the small internal motions of a single protein This technique, diffracted X-ray tracking (DXT)[6], was used to successfully monitor the dynamic twisting motion of KcsA, a pH-sensitive potassium channel, upon gating[6]. We focused on ligand-induced motions of nAChR, using an immobilised gold nanocrystal on the ligand-binding domain of nAChR and on nAChR’s extracellular homologue, AChBP
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