AFM Observation of a Single Receptor Reconstituted into Lipid Bilayer


Nahoko Kasai1 and Chandra S. Ramanujan2
1Materials Science Laboratory, 2University of Oxford

  Ionotropic receptors (ligand-gated ion channel receptors) are important membrane proteins for signaling in the neuronal networks of the central nervous system. They are regulated by a ligand which binds to the extracellular ligand binding domains. This binding gives an allosteric change in the structure and opens the ion channel incorporated in the transmembrane domain to allow cations flow into the cell (Fig. 1). Atomic force microscopy (AFM) enables the nano-scale observation of proteins in a liquid environment, offering a unique opportunity to observe functional biological molecule such as single proteins under physiological conditions.
  In this study, we have succeeded in observing the structure of single purified and ionotropic receptor proteins in the solution using the AFM. Receptor proteins were purified from over-expressed insect cells, and then reconstituted into an artificial lipid bilayer by dialysis because the receptors would function as in vivo when reconstituted into the lipid bilayer. We then imaged the reconstituted receptor proteins on a substrate in a buffer solution using AFM. Before reconstitution, receptors were observed at the edge of small lipid patch on mica (Fig.2 (A)), while after the reconstitution, receptor proteins settled in the large lipid domain on mica (Fig. 2(B)) which provides for the structural observation of the single receptor. Then by zooming in on the single receptor protein, we could observe that it consisted of four protrusions suggesting the four subunits of the receptor protein.
  This study demonstrates that AFM can observe functioning single ionotropic receptors, which suggests the possibility of determining a real-time conformational change of a functioning membrane protein using AFM [1].
  This research was supported in part by Bio-nanotechnology IRC in U.K. and by the Strategic International Cooperative Program, Japan Science and Technology Agency (JST).

[1] N. Kasai, et al., Neurosci. Res. 58 (2007) S193.

Fig. 1. Illustration of a receptor.
Fig. 2. AFM images of receptors before (A) and after (B) reconstituted into artificial lipid bilayer (2×2 µm). Black area: mica substrate, gray: lipid bilayer, white dots: receptor proteins.

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