Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Biophys J
2009 Jan 01;961:226-37. doi: 10.1016/j.bpj.2008.09.034.
Show Gene links
Show Anatomy links
Single-molecule imaging of a fluorescent unnatural amino acid incorporated into nicotinic receptors.
Pantoja R, Rodriguez EA, Dibas MI, Dougherty DA, Lester HA.
???displayArticle.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 Alexa488 (alphaBtxAlexa488). The nAChR has two alphaBtx binding sites, and puncta containing the Lys(BODIPYFL) labeled with alphaBtxAlexa488 yielded the expected three discrete photobleaching steps. We also performed positive control experiments with a nAChR containing enhanced green fluorescent protein in the gamma-subunit M3-M4 loop, which confirmed our nAChR beta19'Lys(BODIPYFL) measurements. Thus, we report on the cell-based single-molecule detection of nAChR beta19'Lys(BODIPYFL).
Anderson,
Fluorescence resonance energy transfer between unnatural amino acids in a structurally modified dihydrofolate reductase.
2002, Pubmed
Anderson,
Fluorescence resonance energy transfer between unnatural amino acids in a structurally modified dihydrofolate reductase.
2002,
Pubmed Baird,
Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral.
2000,
Pubmed Beatty,
Selective dye-labeling of newly synthesized proteins in bacterial cells.
2005,
Pubmed Beatty,
Fluorescence visualization of newly synthesized proteins in mammalian cells.
2006,
Pubmed Betzig,
Imaging intracellular fluorescent proteins at nanometer resolution.
2006,
Pubmed Cha,
Characterizing voltage-dependent conformational changes in the Shaker K+ channel with fluorescence.
1997,
Pubmed
,
Xenbase Charnet,
An open-channel blocker interacts with adjacent turns of alpha-helices in the nicotinic acetylcholine receptor.
1990,
Pubmed Chen,
Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase.
2005,
Pubmed Chen,
Constitutively active G-protein-gated inwardly rectifying K+ channels in dendrites of hippocampal CA1 pyramidal neurons.
2005,
Pubmed Cohen,
Probing protein electrostatics with a synthetic fluorescent amino acid.
2002,
Pubmed Cornish,
Site-specific incorporation of biophysical probes into proteins.
1994,
Pubmed Dahan,
A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site.
2004,
Pubmed
,
Xenbase Deiters,
Site-specific PEGylation of proteins containing unnatural amino acids.
2004,
Pubmed Demuro,
"Optical patch-clamping": single-channel recording by imaging Ca2+ flux through individual muscle acetylcholine receptor channels.
2005,
Pubmed
,
Xenbase Dougherty,
Unnatural amino acids as probes of protein structure and function.
2000,
Pubmed Franz,
Lanthanide-binding tags as versatile protein coexpression probes.
2003,
Pubmed Gallivan,
Site-specific incorporation of biotinylated amino acids to identify surface-exposed residues in integral membrane proteins.
1997,
Pubmed
,
Xenbase Gensler,
Assembly and clustering of acetylcholine receptors containing GFP-tagged epsilon or gamma subunits: selective targeting to the neuromuscular junction in vivo.
2001,
Pubmed
,
Xenbase George,
Specific labeling of cell surface proteins with chemically diverse compounds.
2004,
Pubmed Griffin,
Specific covalent labeling of recombinant protein molecules inside live cells.
1998,
Pubmed Haupts,
Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy.
1998,
Pubmed Hess,
Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.
2006,
Pubmed Hohsaka,
Incorporation of fluorescently labeled nonnatural amino acids into proteins in an E. coli in vitro translation system.
2003,
Pubmed Huang,
Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.
2008,
Pubmed Kajihara,
FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids.
2006,
Pubmed Krafte,
Expression of functional sodium channels in stage II-III Xenopus oocytes.
1989,
Pubmed
,
Xenbase Lummis,
Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel.
2005,
Pubmed Mamaev,
Cell-free N-terminal protein labeling using initiator suppressor tRNA.
2004,
Pubmed Mannuzzu,
Direct physical measure of conformational rearrangement underlying potassium channel gating.
1996,
Pubmed
,
Xenbase Michalet,
Ultrahigh-resolution colocalization of spectrally separable point-like fluorescent probes.
2001,
Pubmed Miller,
Genetic manipulation of ion channels: a new approach to structure and mechanism.
1989,
Pubmed Mitra,
Dynamics of the acetylcholine receptor pore at the gating transition state.
2005,
Pubmed Miyazawa,
Structure and gating mechanism of the acetylcholine receptor pore.
2003,
Pubmed Moerner,
Single-molecule mountains yield nanoscale cell images.
2006,
Pubmed Nashmi,
Assembly of alpha4beta2 nicotinic acetylcholine receptors assessed with functional fluorescently labeled subunits: effects of localization, trafficking, and nicotine-induced upregulation in clonal mammalian cells and in cultured midbrain neurons.
2003,
Pubmed Nashmi,
Chronic nicotine cell specifically upregulates functional alpha 4* nicotinic receptors: basis for both tolerance in midbrain and enhanced long-term potentiation in perforant path.
2007,
Pubmed Noren,
A general method for site-specific incorporation of unnatural amino acids into proteins.
1989,
Pubmed Nowak,
Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells.
1995,
Pubmed
,
Xenbase Nowak,
In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system.
1998,
Pubmed
,
Xenbase Ormö,
Crystal structure of the Aequorea victoria green fluorescent protein.
1996,
Pubmed Paas,
Pore conformations and gating mechanism of a Cys-loop receptor.
2005,
Pubmed
,
Xenbase Rodriguez,
Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: minimizing misacylation.
2007,
Pubmed
,
Xenbase Rodriguez,
Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 2: evaluating suppression efficiency.
2007,
Pubmed
,
Xenbase Rodriguez,
In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression.
2006,
Pubmed
,
Xenbase Shaner,
A guide to choosing fluorescent proteins.
2005,
Pubmed Slimko,
Codon optimization of Caenorhabditis elegans GluCl ion channel genes for mammalian cells dramatically improves expression levels.
2003,
Pubmed
,
Xenbase Sonnleitner,
Structural rearrangements in single ion channels detected optically in living cells.
2002,
Pubmed
,
Xenbase Summerer,
A genetically encoded fluorescent amino acid.
2006,
Pubmed Thompson,
Precise nanometer localization analysis for individual fluorescent probes.
2002,
Pubmed Tong,
Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel.
2001,
Pubmed
,
Xenbase Tsien,
The green fluorescent protein.
1998,
Pubmed Turcatti,
Probing the structure and function of the tachykinin neurokinin-2 receptor through biosynthetic incorporation of fluorescent amino acids at specific sites.
1996,
Pubmed
,
Xenbase Ulbrich,
Subunit counting in membrane-bound proteins.
2007,
Pubmed
,
Xenbase Unwin,
Refined structure of the nicotinic acetylcholine receptor at 4A resolution.
2005,
Pubmed Vázquez-Ibar,
Engineering a terbium-binding site into an integral membrane protein for luminescence energy transfer.
2002,
Pubmed Wang,
Genetically encoding unnatural amino acids for cellular and neuronal studies.
2007,
Pubmed Wang,
A genetically encoded fluorescent amino acid.
2006,
Pubmed Wilson,
Acetylcholine receptor channel structure in the resting, open, and desensitized states probed with the substituted-cysteine-accessibility method.
2001,
Pubmed
,
Xenbase Woolhead,
Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins.
2004,
Pubmed Yao,
Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution.
2005,
Pubmed Yildiz,
Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization.
2003,
Pubmed Zhang,
Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells.
2004,
Pubmed Zhang,
Contribution of the beta subunit M2 segment to the ion-conducting pathway of the acetylcholine receptor.
1998,
Pubmed
,
Xenbase
