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Biophys J
2009 Jan 01;961:226-37. doi: 10.1016/j.bpj.2008.09.034.
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Single-molecule imaging of a fluorescent unnatural amino acid incorporated into nicotinic receptors.
Pantoja R
,
Rodriguez EA
,
Dibas MI
,
Dougherty DA
,
Lester HA
.
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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,
Constitutively active G-protein-gated inwardly rectifying K+ channels in dendrites of hippocampal CA1 pyramidal neurons.
2005,
Pubmed
Chen,
Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase.
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,
In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression.
2006,
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,
Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: minimizing misacylation.
2007,
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,
A genetically encoded fluorescent amino acid.
2006,
Pubmed
Wang,
Genetically encoding unnatural amino acids for cellular and neuronal studies.
2007,
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
