Single-molecule imaging of a fluorescent unnatural amino acid incorporated into nicotinic receptors.

Biophysical Journal
Rigo PantojaHenry A Lester

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 (alpha/beta19'GGGU/gamma/delta) mRNAs. We measured fluorescence from oocytes expressing nAChR beta19'Lys(BODIPYFL), using time-resolved total internal reflection fluorescence microscopy. Under conditions of relatively low receptor density (<0.1 receptors/microm(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 beta19'Lys(BODIPYFL) receptors were similar to those for wild-type receptors. We also studied nAChR beta19'Lys(BODIPYFL) receptors labeled with alpha-bungarotoxin monoconjugated with Al...Continue Reading

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