Single-Molecule Imaging of a Fluorescent Unnatural Amino Acid Incorporated Into Nicotinic Receptors

Abstract

We report on the first, to our knowledge, successful detection of a fluorescent unnatural amino acid (fUAA), Lys(BODIPYFL), incorporated into a membrane protein (the muscle nicotinic acetylcholine receptor, nAChR) in a living cell. Xenopus oocytes were injected with a frameshift-suppressor tRNA, amino-acylated with Lys(BODIPYFL) and nAChR (α/β19′GGGU/γ/δ) mRNAs. We measured fluorescence from oocytes expressing nAChR β19′Lys(BODIPYFL), using time-resolved total internal reflection fluorescence microscopy. Under conditions of relatively low receptor density (<0.1 receptors/μm^2), we observed puncta with diffraction-limited profiles that were consistent with the point-spread function of our microscope. Furthermore, diffraction-limited puncta displayed step decreases in fluorescence intensity, consistent with single-molecule photobleaching. The puncta densities agreed with macroscopic ACh-induced current densities, showing that the fUAA was incorporated, and that receptors were functional. Dose-response relations for the nAChR β19′Lys(BODIPYFL) receptors were similar to those for wild-type receptors. We also studied nAChR β19′Lys(BODIPYFL) receptors labeled with α-bungarotoxin monoconjugated with Alexa488 (αBtxAlexa488). The nAChR has two αBtx binding sites, and puncta containing the Lys(BODIPYFL) labeled with αBtxAlexa488 yielded the expected three discrete photobleaching steps. We also performed positive control experiments with a nAChR containing enhanced green fluorescent protein in the γ-subunit M3-M4 loop, which confirmed our nAChR β19′Lys(BODIPYFL) measurements. Thus, we report on the cell-based single-molecule detection of nAChR β19′Lys(BODIPYFL).