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

Pantoja, Rigo and Rodriguez, Erik A. and Dibas, Mohammed I. and Dougherty, Dennis A. and Lester, Henry A. (2009) Single-Molecule Imaging of a Fluorescent Unnatural Amino Acid Incorporated Into Nicotinic Receptors. Biophysical Journal, 96 (1). pp. 226-237. ISSN 0006-3495. PMCID PMC2710013. doi:10.1016/j.bpj.2008.09.034. https://resolver.caltech.edu/CaltechAUTHORS:20090909-105237809

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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).


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/j.bpj.2008.09.034DOIArticle
http://www.cell.com/biophysj/abstract/S0006-3495(08)00012-XPublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2710013/PubMed CentralArticle
ORCID:
AuthorORCID
Dougherty, Dennis A.0000-0003-1464-2461
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2009 Biophysical Society. Submitted February 29, 2008, and accepted for publication September 29, 2008. We gratefully acknowledge the assistance of Dr. E. James Petersson at the outset of this project. We thank Dr. Shelly Tzlil for single-molecule discussions, and for help with programming. Mr. Edward Wang provided assistance with ImageJ Java programming and data documentation. We also thank the members of the Lester and Dougherty Laboratory Group for invaluable discussions. R.P. was supported by postdoctoral fellowships from the Ford Foundation and American Psychological Association Diversity Program in Neuroscience. M.I.D. was supported by a National Research Service Award from the National Institutes of Health. E.A.R. is a National Science Foundation Predoctoral Fellow. This work was supported by National Institutes of Health grants NS 11756, NS 34407, and HL 79350. Two figures are available at http://www.biophysj.org/biophysj/supplemental/S0006-3495(08)00012-X.
Funders:
Funding AgencyGrant Number
Ford FoundationUNSPECIFIED
NIHNS 11756
NIHNS 34407
NIHHL 79350
American Psychological AssociationUNSPECIFIED
NSFUNSPECIFIED
Issue or Number:1
PubMed Central ID:PMC2710013
DOI:10.1016/j.bpj.2008.09.034
Record Number:CaltechAUTHORS:20090909-105237809
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20090909-105237809
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:15688
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:10 Sep 2009 18:14
Last Modified:08 Nov 2021 23:21

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