Research on the regulation of the spatial structure of acetylcholinesterase tetramer with high efficiency by AFM

Abstract

Atomic force microscopy (AFM) was applied for obtaining structural information about acetylcholinesterase (AChE) tetramer (AChE G(4)) before and after reaction with S-acetylcholine iodide (S-ACh), in the presence or absence of propidium iodide (PI), an inhibitor for peripheral anionic sites (PAS). An iced-bath ultrasound was used to prepare the phospholipid membrane. Ves-fusion technique was applied for incorporating AChE G(4) in a lipid layer on mica. Before reaction with substrates, the single AChE G(4) particle was ellipsoid in shape with a clear border. It had a smooth surface with a central projection. The four subunits of a single enzyme particle were arranged tightly (no separated subunits being found, with an average size of 89 ± 7 nm in length, 68 ± 9 nm in width, and 6 ± 3 nm in height). After reaction with S-ACh in the absence of PI, the loose arrangement of subunits of AChE G(4) was seen, with an average size of 104 ± 7 nm in length, 91 ± 5 nm in width, and 8 ± 2 nm in height. Also there was free-flowing space amongst the four subunits of the AChE G(4). This was consistent with the results of the ×-ray diffraction crystallography and molecular dynamics studies. The apparent free space was the central path of AChE G(4), changing from small to big, to small, to lateral door appearance, with an average size of 60 ± 5 nm in length and 51 ± 9 nm in width. The size of lateral door was 52 ± 5 nm in width and 32 ± 3 nm in depth on average. In the presence of PI, S-ACh could not cause topological structure changes of AChE G(4). AFM verified that the central path might govern the turnover of the enzyme morphologically, and the interactions between PI and S-ACh might gate the creation of a central path and the opening of ACG in monomer; and the combination of S-ACh with peripheral anionic sites is conducive to the opening of ACG while PI can inhibit this action. Resolution at the inframolecular level is favorable in providing substantial information on how the spatial structure is adapted to the high efficiency of AChE molecules.

Keywords: ACG; AChE; AFM; Ves-fusion; phospholipid membrane.

Figure 1

AFM images presented in contact mode of phospholipid membrane reconstituted on mica. (A) Large-scale view of a phospholipid membrane. (B) Higher magnification view showing a region of fattened membrane with some pore spaces. Notes: Light region is phospholipid membrane, and dark region is surface of exposed mica. The fusion of the membrane is perfect; not seeing apparent defects. Lines AB, CD, and EF are examples of AFM topographic line profiles of the membrane. The measurements of the height difference between the rim of the membrane show that the height of the phospholipid membrane is on average 2.26 nm (n = 30), which is the thickness of a lipid’s single layer. Abbreviations: AFM, atomic force microscopy; deg, degree; ìm, micrometers; nm, nanometers.

Figure 2

AFM images presented in the tapping mode of AChE g₄ incorporated in a lipid layer on mica. Images are collected by (A) height mode and (B) phase mode, simultaneously. The same AChE g₄ particle incorporated in a lipid layer is indicated by white arrows, and can be seen clearly in (A and B). From (B), we can see some minute information, including the rim of protein reconstituted into membranes, which is highlighted, suggesting that there are some interactions between proteins and lipids. There are borders between the membranes, indicated by the white rectangle and circle, which are both not seen from the height map (A). Note: All image sizes are 1.87 μm × 1.87 μm. Abbreviations: AFM, atomic force microscopy; AChE g₄, acetylcholinesterase tetramer; ìm, micrometers; nm, nanometers; v, volts.

Figure 3

Comparisons of AFM images of AChE G₄ incorporated in a lipid layer on mica (A) before reaction with substrates, and (B–E) after reaction with S-ACh in the absence or (F) presence of PI, an inhibitor for peripheral anionic sites. The loose arrangement of the subunits of AChE g₄ is the most obvious effect followed by S-ACh (B–E), and the volume of AChE g₄ after reaction with S-ACh is larger than (A), and there is an apparent free space in the center of AChE g₄, changing from (B) small to (C) big, to (D) small, to (E) a lateral door appearance. (F) In the presence of a PAS inhibitor, S-ACh could not cause topological structure changes of AChE g₄. Note: All image sizes are 1.87 μm × 1.87 μm. Abbreviations: AFM, atomic force microscopy; AChE g₄, acetylcholinesterase tetramer; ACh, acetylcholine; μm, micrometers; nm, nanometers; S-ACh, S-acetylcholine iodide; PI, propidium iodide; PAS, peripheral anionic sites.

Figure 4

Comparisons of high-resolution AFM images of AChE G₄ incorporated in a lipid layer on mica (A) before and (C and E) after reaction with S-ACh in the absence or (G) presence of PI, an inhibitor for peripheral anionic sites. (A, C, E and G) are the magnification of the proteins circled in (Figure 3A, D, E and F), respectively, and (B, D, F, and H) are their corresponding high-resolution three-dimensional images. (A and B) A single AChE g₄ particle before reaction with S-ACh is ellipsoid and smooth with a center projection and clear border. (G and H) In the presence of PI, the topological structure of AChE g₄ has not changed. Note: All image sizes are 300.62 nm × 300.62 nm. Abbreviations: AFM, atomic force microscopy; AChE g₄, acetylcholinesterase tetramer; nm, nanometers; S-ACh, S-acetylthiocholine iodide; PI, propidium iodide.