XB-ART-49181
Chembiochem
2014 Aug 18;1512:1710-20. doi: 10.1002/cbic.201402080.
Show Gene links
Show Anatomy links
In vivo incorporation of non-canonical amino acids by using the chemical aminoacylation strategy: a broadly applicable mechanistic tool.
Dougherty DA
,
Van Arnam EB
.
???displayArticle.abstract???
We describe a strategy for incorporating non-canonical amino acids site-specifically into proteins expressed in living cells, involving organic synthesis to chemically aminoacylate a suppressor tRNA, protein expression in Xenopus oocytes, and monitoring protein function, primarily by electrophysiology. With this protocol, a very wide range of non-canonical amino acids can be employed, allowing both systematic structure-function studies and the incorporation of reactive functionalities. Here, we present an overview of the methodology and examples meant to illustrate the versatility and power of the method as a tool for investigating protein structure and function.
???displayArticle.pubmedLink??? 24990307
???displayArticle.pmcLink??? PMC4155927
???displayArticle.link??? Chembiochem
???displayArticle.grants??? [+]
Genes referenced: mt-tr trna
References [+] :
Ahern,
Electrostatic contributions of aromatic residues in the local anesthetic receptor of voltage-gated sodium channels.
2008, Pubmed,
Xenbase
Ahern, Electrostatic contributions of aromatic residues in the local anesthetic receptor of voltage-gated sodium channels. 2008, Pubmed , Xenbase
Ahern, A cation-pi interaction between extracellular TEA and an aromatic residue in potassium channels. 2006, Pubmed
Ahern, An electrostatic interaction between TEA and an introduced pore aromatic drives spring-in-the-door inactivation in Shaker potassium channels. 2009, Pubmed , Xenbase
Baldini, Mischarging Escherichia coli tRNAPhe with L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine, a photoactivatable analogue of phenylalanine. 1988, Pubmed
Beene, Unnatural amino acid mutagenesis in mapping ion channel function. 2003, Pubmed , Xenbase
Blum, Evidence for an extended hydrogen bond network in the binding site of the nicotinic receptor: role of the vicinal disulfide of the alpha1 subunit. 2011, Pubmed , Xenbase
Blum, Nicotinic pharmacophore: the pyridine N of nicotine and carbonyl of acetylcholine hydrogen bond across a subunit interface to a backbone NH. 2010, Pubmed , Xenbase
Cashin, Chemical-scale studies on the role of a conserved aspartate in preorganizing the agonist binding site of the nicotinic acetylcholine receptor. 2007, Pubmed , Xenbase
CHAPEVILLE, On the role of soluble ribonucleic acid in coding for amino acids. 1962, Pubmed
Chen, Detection of dihydrofolate reductase conformational change by FRET using two fluorescent amino acids. 2013, Pubmed
Cohen, Probing protein electrostatics with a synthetic fluorescent amino acid. 2002, Pubmed
Corringer, Structure and pharmacology of pentameric receptor channels: from bacteria to brain. 2012, Pubmed
Corringer, Atomic structure and dynamics of pentameric ligand-gated ion channels: new insight from bacterial homologues. 2010, Pubmed
Dang, Probing the role of a conserved M1 proline residue in 5-hydroxytryptamine(3) receptor gating. 2000, Pubmed , Xenbase
Deechongkit, Context-dependent contributions of backbone hydrogen bonding to beta-sheet folding energetics. 2004, Pubmed
Dougherty, Cation-pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. 1996, Pubmed
Dougherty, Acetylcholine binding by a synthetic receptor: implications for biological recognition. 1990, Pubmed
Dougherty, The cation-π interaction. 2013, Pubmed
Dougherty, Cys-loop neuroreceptors: structure to the rescue? 2008, Pubmed
Duffy, Preparation of translationally competent tRNA by direct chemical acylation. 2010, Pubmed , Xenbase
Eastwood, A selenide-based approach to photochemical cleavage of peptide and protein backbones at engineered backbone esters. 2009, Pubmed
England, Site-specific, photochemical proteolysis applied to ion channels in vivo. 1997, Pubmed , Xenbase
England, Mapping disulfide connectivity using backbone ester hydrolysis. 1999, Pubmed , Xenbase
England, Backbone mutations in transmembrane domains of a ligand-gated ion channel: implications for the mechanism of gating. 1999, Pubmed , Xenbase
Fahmi, Site-specific incorporation of glycosylated serine and tyrosine derivatives into proteins. 2007, Pubmed
Gallivan, Site-specific incorporation of biotinylated amino acids to identify surface-exposed residues in integral membrane proteins. 1997, Pubmed , Xenbase
Gleitsman, An intersubunit hydrogen bond in the nicotinic acetylcholine receptor that contributes to channel gating. 2008, Pubmed
Gleitsman, Probing the role of backbone hydrogen bonding in a critical beta sheet of the extracellular domain of a cys-loop receptor. 2009, Pubmed
Goto, Flexizymes for genetic code reprogramming. 2011, Pubmed
Guo, Addition of an alpha-hydroxy acid to the genetic code of bacteria. 2008, Pubmed
Hanek, Photochemical proteolysis of an unstructured linker of the GABAAR extracellular domain prevents GABA but not pentobarbital activation. 2010, Pubmed
Hanek, A stereochemical test of a proposed structural feature of the nicotinic acetylcholine receptor. 2008, Pubmed
Hartman, Enzymatic aminoacylation of tRNA with unnatural amino acids. 2006, Pubmed
Hartman, An expanded set of amino acid analogs for the ribosomal translation of unnatural peptides. 2007, Pubmed
Hashimoto, Simple and quick chemical aminoacylation of tRNA in cationic micellar solution under ultrasonic agitation. 2005, Pubmed
Hecht, "Chemical aminoacylation" of tRNA's. 1978, Pubmed
Heckler, T4 RNA ligase mediated preparation of novel "chemically misacylated" tRNAPheS. 1984, Pubmed
Johnson, Residue-specific incorporation of non-canonical amino acids into proteins: recent developments and applications. 2010, Pubmed
Kearney, Determinants of nicotinic receptor gating in natural and unnatural side chain structures at the M2 9' position. 1996, Pubmed , Xenbase
Koh, An experimental approach to evaluating the role of backbone interactions in proteins using unnatural amino acid mutagenesis. 1997, Pubmed
Köhrer, Import of amber and ochre suppressor tRNAs into mammalian cells: a general approach to site-specific insertion of amino acid analogues into proteins. 2001, Pubmed
Lester, Heterologous expression of excitability proteins: route to more specific drugs? 1988, Pubmed
Li, The tethered agonist approach to mapping ion channel proteins--toward a structural model for the agonist binding site of the nicotinic acetylcholine receptor. 2001, Pubmed , Xenbase
Limapichat, Chemical scale studies of the Phe-Pro conserved motif in the cys loop of Cys loop receptors. 2010, Pubmed , Xenbase
Link, Reassignment of sense codons in vivo. 2005, Pubmed
Link, Non-canonical amino acids in protein engineering. 2003, Pubmed
Lu, Probing ion permeation and gating in a K+ channel with backbone mutations in the selectivity filter. 2001, Pubmed , Xenbase
Lummis, Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel. 2005, Pubmed
Ma, The Cationminus signpi Interaction. 1997, Pubmed
McMenimen, Probing the Mg2+ blockade site of an N-methyl-D-aspartate (NMDA) receptor with unnatural amino acid mutagenesis. 2006, Pubmed , Xenbase
Miller, Flash decaging of tyrosine sidechains in an ion channel. 1998, Pubmed , Xenbase
Monahan, Site-specific incorporation of unnatural amino acids into receptors expressed in Mammalian cells. 2003, Pubmed
Neher, Single-channel currents recorded from membrane of denervated frog muscle fibres. 1976, Pubmed
Noren, A general method for site-specific incorporation of unnatural amino acids into proteins. 1989, Pubmed
Normanly, tRNA identity. 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
Padgett, Unnatural amino acid mutagenesis of the GABA(A) receptor binding site residues reveals a novel cation-pi interaction between GABA and beta 2Tyr97. 2007, Pubmed , Xenbase
Pantoja, Single-molecule imaging of a fluorescent unnatural amino acid incorporated into nicotinic receptors. 2009, Pubmed , Xenbase
Petersson, MALDI-TOF mass spectrometry methods for evaluation of in vitro aminoacyl tRNA production. 2002, Pubmed
Petersson, A perturbed pK(a) at the binding site of the nicotinic acetylcholine receptor: implications for nicotine binding. 2002, Pubmed , Xenbase
Petersson, Caging proteins through unnatural amino acid mutagenesis. 2003, Pubmed , Xenbase
Philipson, Incorporation of caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor. 2001, Pubmed , Xenbase
Pless, Molecular basis for class Ib anti-arrhythmic inhibition of cardiac sodium channels. 2011, Pubmed
Pless, Unnatural amino acids as probes of ligand-receptor interactions and their conformational consequences. 2013, Pubmed
Price, A hydrogen bond in loop A is critical for the binding and function of the 5-HT3 receptor. 2008, Pubmed
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
Saks, An engineered Tetrahymena tRNAGln for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression. 1996, Pubmed , Xenbase
Santarelli, Calcium block of single sodium channels: role of a pore-lining aromatic residue. 2007, Pubmed
Santarelli, A cation-pi interaction discriminates among sodium channels that are either sensitive or resistant to tetrodotoxin block. 2007, Pubmed , Xenbase
Sisido, Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids. 2005, Pubmed
Tavares, Variations in binding among several agonists at two stoichiometries of the neuronal, α4β2 nicotinic receptor. 2012, Pubmed
Tong, Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel. 2001, Pubmed , Xenbase
Torrice, Probing the role of the cation-pi interaction in the binding sites of GPCRs using unnatural amino acids. 2009, Pubmed , Xenbase
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
Van Arnam, Dissecting the functions of conserved prolines within transmembrane helices of the D2 dopamine receptor. 2011, Pubmed , Xenbase
Xiu, Nicotine binding to brain receptors requires a strong cation-pi interaction. 2009, Pubmed , Xenbase
Young, Beyond the canonical 20 amino acids: expanding the genetic lexicon. 2010, Pubmed
Zacharias, Cation-pi interactions in ligand recognition and catalysis. 2002, Pubmed
Zhong, From ab initio quantum mechanics to molecular neurobiology: a cation-pi binding site in the nicotinic receptor. 1998, Pubmed