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In Vivo Studies of Receptors and Ion Channels with Unnatural Amino Acids

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Protein Engineering

Part of the book series: Nucleic Acids and Molecular Biology ((NUCLEIC,volume 22))

The combination of nonsense suppression for unnatural amino acid incorporation and heterologous expression in Xenopus oocytes provides a powerful means to probe neuroreceptors and ion channels with chemical precision. Here we describe a range of studies that illustrate the broad potential of this approach. A variety of biophysical probes and reactive moieties can be incorporated. In addition, subtle systematic variations allow detailed, physical organic chemistry studies of the complex proteins of neuroscience.

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References

  • Ahern CA, Eastwood AL, Lester HA, Dougherty DA, Horn R (2006) A cation—π interaction between extracellular TEA and an aromatic residue in potassium channels. J Gen Physiol 128:649–657

    Article  PubMed  CAS  Google Scholar 

  • Anderson RD, Zhou J, Hecht SM (2002) Fluorescence resonance energy transfer between unnatural amino acids in a structurally modified dihydrofolate reductase. J Am Chem Soc 124: 9674–9675

    Article  PubMed  CAS  Google Scholar 

  • Bain JD, Glabe CG, Dix TA, Chamberlin AR (1989) Biosynthetic site-specific incorporation of a non-natural amino acid into a polypeptide. J Am Chem Soc 111:8013–8014

    Article  CAS  Google Scholar 

  • Beene DL, Brandt GS, Zhong WG, Zacharias NM, Lester HA, Dougherty DA (2002) Cation—pi interactions in ligand recognition by serotonergic (5-HT3A) and nicotinic acetylcholine receptors: the anomalous binding properties of nicotine. Biochemistry 41:10262–10269

    Article  PubMed  CAS  Google Scholar 

  • Beene DL, Dougherty DA, Lester HA (2003) Unnatural amino acid mutagenesis in mapping ion channel function. Curr Opin Neurobiol 13:264–270

    Article  PubMed  CAS  Google Scholar 

  • Beene DL, Price KL, Lester HA, Dougherty DA, Lummis SCR (2004) Tyrosine residues that control binding and gating in the 5-hydroxytryptamine(3) receptor revealed by unnatural amino acid mutagenesis. J Neurosci 24:9097–9104

    Article  PubMed  CAS  Google Scholar 

  • Cashin AL, Petersson EJ, Lester HA, Dougherty DA (2005) Using physical chemistry to differentiate nicotinic from cholinergic agonists at the nicotinic acetylcholine receptor. J Am Chem Soc 127:350–356

    Article  PubMed  CAS  Google Scholar 

  • Cashin AL, Torrice MM, McMenimen KA, Lester HA, Dougherty DA (2007) Chemical-scale studies on the role of a conserved aspartate in preorganizing the agonist binding site of the nicotinic acetylcholine receptor. Biochemistry 46:630–639

    Article  PubMed  CAS  Google Scholar 

  • Cha A, Snyder GE, Selvin PR, Bezanilla F (1999) Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy. Nature 402:809–813

    Article  PubMed  CAS  Google Scholar 

  • Cohen BE, McAnaney TB, Park ES, Jan YN, Boxer SG, Jan LY (2002) Probing protein electrostatics with a synthetic fluorescent amino acid. Science 296:1700–1703

    Article  PubMed  CAS  Google Scholar 

  • Cornish VW, Benson DR, Altenbach CA, Hideg K, Hubbell WL, Schultz PG (1994) Site-specific incorporation of biophysical probes into proteins. Proc Natl Acad Sci USA 91:2910–2914

    Article  PubMed  CAS  Google Scholar 

  • Cornish VW, Mendel D, Schultz PG (1995) Probing protein structure and function with an expanded genetic code. Angew Chem Int Ed Engl 34:621–633

    Article  CAS  Google Scholar 

  • Cornish VW, Hahn KM, Schultz PG (1996) Site specific protein modification using a ketone handle. J Am Chem Soc 118:8150–8151

    Article  CAS  Google Scholar 

  • Corringer P-J, Le Novère N, Changeux J-P (2000) Nicotinic receptors at the amino acid level. Annu Rev Pharmacol Toxicol 40:431–458

    Article  PubMed  CAS  Google Scholar 

  • Dahan DS, Dibas MI, Petersson EJ, Auyeung VC, Chanda B, Bezanilla F, Dougherty DA, Lester HA (2004) A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site. Proc Natl Acad Sci USA 101:10195–10200

    Article  PubMed  CAS  Google Scholar 

  • Dang H, England PM, Sarah Farivar S, Dougherty DA, Lester HA (2000) Probing the role of a conserved M1 proline residue in 5-hydroxytryptamine3 receptor gating. Mol Pharm 57:1114–1122

    CAS  Google Scholar 

  • Dieterich DC, Link AJ, Graumann J, Tirrell DA, Schuman EM (2006) Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc Natl Acad Sci USA 103:9482–9487

    Article  PubMed  CAS  Google Scholar 

  • Dougherty DA (1996) Cation—π interactions in chemistry and biology. a new view of benzene, Phe, Tyr, and Trp. Science 271:163–168

    Article  PubMed  CAS  Google Scholar 

  • Dougherty DA (2000) Unnatural amino acids as probes of protein structure and function. Curr Opin Chem Biol 4:645–652

    Article  PubMed  CAS  Google Scholar 

  • Dougherty DA, Stauffer DA (1990) Acetylcholine binding by a synthetic receptor. Implications for biological recognition. Science 250:1558–1560

    Article  PubMed  CAS  Google Scholar 

  • England PM, Lester HA, Davidson N, Dougherty DA (1997) Site-specific, photochemical proteolysis applied to ion channels in vivo. Proc Natl Acad Sci USA 94:11025–11030

    Article  PubMed  CAS  Google Scholar 

  • England PM, Lester HA, Dougherty DA (1999a) Mapping disulfide connectivity using backbone ester hydrolysis. Biochemistry 38:14409–14415

    Article  CAS  Google Scholar 

  • England PM, Zhang Y, Dougherty DA, Lester HA (1999b) Backbone mutations in transmembrane domains of a ligand-gated ion channel: implications for the mechanism of gating. Cell 96:89–98

    Article  CAS  Google Scholar 

  • Fahnesto S, Neumann H, Shashoua V, Rich A (1970) Ribosome-catalyzed ester formation. Biochemistry 9:2477–2478

    Article  Google Scholar 

  • Gallivan JP, Lester HA, Dougherty DA (1997) Site-specific incorporation of biotinylated amino acids to identify surface-exposed residues in integral membrane proteins. Chem Biol 4: 739–749

    Article  PubMed  CAS  Google Scholar 

  • Glauner KS, Mannuzzu LM, Gandhi CS, Isacoff EY (1999) Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel. Nature 402:813–817

    Article  PubMed  CAS  Google Scholar 

  • Heckler TG, Chang L-H, Zama Y, Naka T, Chorghade MS, Hecht SM (1984) T4 RNA ligase mediated preparation of novel “chemically misacylated” tRNAPhes. Biochemistry 23: 1468–1473

    Article  PubMed  CAS  Google Scholar 

  • Hohsaka T, Ashizuka Y, Murakami H, Sisido M (1996) Incorporation of nonnatural amino acids into streptavidin through in vitro frame-shift suppression. J Am Chem Soc 118:9778–9779

    Article  CAS  Google Scholar 

  • Hubbell WL, Altenbach C, Hubbell CM, Khorana HG (2003) Rhodopsin structure, dynamics, and activation: a perspective from crystallography, site-directed spin labeling, sulfhydryl reactivity, and disulfide cross-linking. In: Membrane proteins, vol 63: advances in protein chemistry, pp 243–290

    Article  CAS  Google Scholar 

  • Ilegems E, Pick HM, Vogel H (2002) Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery. Nucleic Acids Res 30:e128

    Google Scholar 

  • Karlin A (2002) Emerging structure of the nicotinic acetylcholine receptors. Nat Rev Neurosci 3:102–114

    Article  PubMed  CAS  Google Scholar 

  • Kearney P, Nowak M, Zhong W, Silverman S, Lester H, Dougherty D (1996a) Dose-response relations for unnatural amino acids at the agonist binding site of the nicotinic acetylcholine receptor: tests with novel side chains and with several agonists. Mol Pharmacol 50:1401–1412

    CAS  Google Scholar 

  • Kearney PC, Zhang H, Zhong W, Dougherty DA, Lester HA (1996b) Determinants of nicotinic receptor gating in natural and unnatural side chain structures at the M2 9′ position. Neuron 17:1221–1229

    Article  CAS  Google Scholar 

  • Koh JT, Cornish VW, Schultz PG (1997) An experimental approach to evaluating the role of backbone interactions in proteins using unnatural amino acid mutagenesis. Biochemistry 36:11314–11322

    Article  PubMed  CAS  Google Scholar 

  • Labarca C, Nowak MW, Zhang H, Tang L, Deshpande P, Lester HA (1995) Channel gating governed symmetrically by conserved leucine residues in the M2 domain of nicotinic receptors. Nature 376:514–516

    Article  PubMed  CAS  Google Scholar 

  • Lester HA, Dibas MI, Dahan DS, Leite JF, Dougherty DA (2004) Cys-loop receptors: new twists and turns. Trends Neurosci 27:329–336

    Article  PubMed  CAS  Google Scholar 

  • Li LT, Zhong WG, Zacharias N, Gibbs C, Lester HA, Dougherty DA (2001) The tethered agonist approach to mapping ion channel proteins toward a structural model for the agonist binding site of the nicotinic acetylcholine receptor. Chem Biol 8:47–58

    Article  PubMed  Google Scholar 

  • Link AJ, Tirrell DA (2005) Reassignment of sense codons in vivo. Methods 36:291–298

    Article  PubMed  CAS  Google Scholar 

  • Long SB, Campbell EB, MacKinnon R (2005) Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309:897–903

    Article  PubMed  CAS  Google Scholar 

  • Lummis SCR, Beene DL, Harrison NJ, Lester HA, Dougherty DA (2005a) A cation—pi binding interaction with a tyrosine in the binding site of the GABA(C) receptor. Chem Biol 12:993–997

    Article  CAS  Google Scholar 

  • Lummis SCR, Beene DL, Lee LW, Lester HA, Broadhurst RW, Dougherty DA (2005b) Cis—trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel. Nature 438:248–252

    Article  CAS  Google Scholar 

  • Ma JC, Dougherty DA (1997) The cation—π interaction. Chem Rev 97:1303–1324

    Article  PubMed  CAS  Google Scholar 

  • McMenimen KA, Petersson EJ, Lester HA, Dougherty DA (2006) Probing the Mg2+ blockade site of an N-methyl-d-aspartate (NMDA) receptor with unnatural amino acid mutagenesis. Acs Chem Biol 1:227–234

    Article  PubMed  CAS  Google Scholar 

  • Mecozzi S, West Jr AP, Dougherty DA (1996a) Cation—π Interactions in aromatics of biological and medicinal interest: electrostatic potential surfaces as a useful qualitative guide. Proc Natl Acad Sci USA 93:10566–10571

    Article  CAS  Google Scholar 

  • Mecozzi S, West Jr AP, Dougherty DA (1996b) Cation—π interactions in simple aromatics. Electrostatics provide a predictive tool. J Am Chem Soc 118:2307–2308

    Article  CAS  Google Scholar 

  • Miller JC, Silverman SK, England PM, Dougherty DA, Lester HA (1998) Flash decaging of tyrosine sidechains in an ion channel. Neuron 20:619–624.

    Article  PubMed  CAS  Google Scholar 

  • Monahan SL, Lester HA, Dougherty DA (2003) Site-specific incorporation of unnatural amino acids into receptors expressed in mammalian cells. Chem Biol 10:573–580

    Article  PubMed  CAS  Google Scholar 

  • Mu TW, Lester HA, Dougherty DA (2003) Different binding orientations for the same agonist at homologous receptors: a lock and key or a simple wedge? J Am Chem Soc 125:6850–6851

    Article  PubMed  CAS  Google Scholar 

  • Neher E, Sakmann B (1976) Single channels recorded from membrane of denervated frog muscle fibres. Nature 260:799–802

    Article  PubMed  CAS  Google Scholar 

  • Noren CJ, Anthony-Cahill SJ, Griffith MC, Schultz PG (1989) A general-method for site-specific incorporation of unnatural amino-acids into proteins. Science 244:182–188

    Article  PubMed  CAS  Google Scholar 

  • Nowak MW, Kearney PC, Sampson JR, Saks ME, Labarca CG, Silverman SK, Zhong W, Thorson J, Abelson JN, Davidson N, Schultz PG, Dougherty DA, Lester HA (1995) Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells. Science 268: 439–442

    Article  PubMed  CAS  Google Scholar 

  • Nowak MW, Gallivan JP, Silverman SK, Labarca CG, Dougherty DA, Lester HA (1998) In vivo incorporation of unnatural amino acids into ion channels in a Xenopus oocyte expression system. Methods Enzymol 293:504–529

    Article  PubMed  CAS  Google Scholar 

  • Padgett CL, Hanek AP, Lester HA, Dougherty DA, Lummis SC (2007) Unnatural amino acid mutagenesis of the GABA(A) receptor binding site residues reveals a novel cation—π interaction between GABA and β2Tyr97. J Neurosci 27:886–892

    Article  PubMed  CAS  Google Scholar 

  • Perozo E (2006) Gating prokaryotic mechanosensitive channels. Nat Rev Mol Cell Biol 7: 109–119

    Article  PubMed  CAS  Google Scholar 

  • Petersson EJ, Choi A, Dahan DS, Lester HA, Dougherty DA (2002) A perturbed pK(a) at the binding site of the nicotinic acetylcholine receptor: implications for nicotine binding. J Am Chem Soc 124:12662–12663

    Article  PubMed  CAS  Google Scholar 

  • Petersson EJ, Brandt GS, Zacharias NM, Dougherty DA, Lester HA (2003) Caging proteins through unnatural amino acid mutagenesis. Methods Enzymol 360:258–273

    Article  PubMed  CAS  Google Scholar 

  • Philipson KD, Gallivan JP, Brandt GS, Dougherty DA, Lester HA (2001) Incorporation of caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor. Am J Physiol Cell Physiol 281:195–206

    Google Scholar 

  • Prescher JA, Bertozzi CR (2005) Chemistry in living systems. Nat Chem Biol 1:13–21

    Article  PubMed  CAS  Google Scholar 

  • Price KL, Beene DL, Dougherty DA, Lester HA, Lummis SCR (2003) The role of tyrosine residues at the mouse 5-HT3A receptor ligand binding site investigated by unnatural amino acid mutagenesis. Brit J Pharmacol 140

    Google Scholar 

  • Revah F, Bertrand D, Galzi JL, Devillers-Theiry A, Mulle C (1991) Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor. Nature 353:846–849

    Article  PubMed  CAS  Google Scholar 

  • Rodriguez EA, Lester HA, Dougherty DA (2006) In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression. Proc Natl Acad Sci USA 103: 8650–8655

    Article  PubMed  CAS  Google Scholar 

  • Rodriguez EA, Lester HA, Dougherty DA (2007a) Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo, part 1: minimizing misacylation. RNA 13: 1703–1714

    Article  CAS  Google Scholar 

  • Rodriguez EA, Lester HA, Dougherty DA (2007b) Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo, part 2: evaluating suppression efficiency. RNA 13:1715–1722

    Article  CAS  Google Scholar 

  • Sakamoto K, Hayashi A, Sakamoto A, Kiga D, Nakayama H, Soma A, Kobayashi T, Kitabatake M, Takio K, Saito K, Shirouzu M, Hirao I, Yokoyama S (2002) Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells. Nucleic Acids Res 30:4692–4699

    Article  PubMed  CAS  Google Scholar 

  • Saks ME, Sampson JR, Nowak MW, Kearney PC, Du F, Abelson JN, Lester HA, Dougherty DA (1996) An engineered Tetrahymena tRNAGln for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression. J Biol Chem 271:23169–23175

    Article  PubMed  CAS  Google Scholar 

  • Santarelli VP, Eastwood AL, Dougherty DA, Horn R, Ahern CA (2007) A cation—π interaction discriminates among sodium channels that are either sensitive or resistant to tetrodotoxin block. J Biol Chem 282:8044–8051

    Article  PubMed  CAS  Google Scholar 

  • Shafer AM, Kalai T, Liu SQB, Hideg K, Voss JC (2004) Site-specific insertion of spin-labeled l-amino acids in Xenopus oocytes. Biochemistry 43:8470–8482

    Article  PubMed  CAS  Google Scholar 

  • Summerer D, Chen S, Wu N, Deiters A, Chin JW, Schultz PG (2006) A genetically encoded fluorescent amino acid. Proc Natl Acad Sci USA 103:9785–9789

    Article  PubMed  CAS  Google Scholar 

  • Taki M, Hohsaka T, Murakami H, Taira K, Sisido M (2002) Position-specific incorporation of a fluorophore-quencher pair into a single streptavidin through orthogonal four-base codon/anti-codon pairs. J Am Chem Soc 124:14586–14590

    Article  PubMed  CAS  Google Scholar 

  • Tong Y, Brandt GS, Li M, Shapovalov G, Slimko E, Karschin A, Dougherty DA, Lester HA (2001) Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel. J Gen Physiol 117:103–118

    Article  PubMed  CAS  Google Scholar 

  • Turcatti G, Nemeth K, Edgerton MD, Meseth U, Talabot F, Peitsch M, Knowles J, Vogel H, Chollet A (1996) Probing the structure and function of the tachykinin neurokinin-2 receptor through biosynthetic incorporation of fluorescent amino acids at specific sites. J Biol Chem 271:19991–19998

    Article  PubMed  CAS  Google Scholar 

  • Turcatti G, Nemeth K, Edgerton MD, Knowles J, Vogel H, Chollet A (1997) Fluorescent labeling of NK2 receptor at specific sites in vivo and fluorescence energy transfer analysis of NK2 ligandreceptor complexes. Receptors Channels 5:201–207

    PubMed  CAS  Google Scholar 

  • Unwin N (2005) Refined structure of the nicotinic acetylcholin receptor at 4 A resolution. J Mol Biol 346:967–989

    Article  PubMed  CAS  Google Scholar 

  • van Swieten PF, Leeuwenburgh MA, Kessler BM, Overkleeft HS (2005) Bioorthogonal organic chemistry in living cells: Novel strategies for labeling biomolecules. Org Biomol Chem 3: 20–27

    Article  PubMed  CAS  Google Scholar 

  • Wang L, Schultz PG (2005) Expanding the genetic code. Angew Chem Int Ed Engl 44:34–66

    Article  CAS  Google Scholar 

  • Wang JY, Xie JM, Schultz PG (2006) A genetically encoded fluorescent amino acid. J Am Chem Soc 128:8738–8739

    Article  PubMed  CAS  Google Scholar 

  • Wilson GG, Karlin A (1998) The location of the gate in the acetylcholine receptor channel. Neuron 20:1269–1281

    Article  PubMed  CAS  Google Scholar 

  • Zhong W, Gallivan JP, Zhang Y, Li L, Lester HA, Dougherty DA (1998) From ab initio quantum mechanics to molecular neurobiology: A cation—π binding site in the nicotinic receptor. Proc Natl Acad Sci USA 95:12088–12093

    Article  PubMed  CAS  Google Scholar 

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  1. Division of Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, CA, 91125, USA

    D. A. Dougherty

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  1. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA

    Caroline Köhrer  & Uttam L. RajBhandary  & 

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Dougherty, D.A. (2009). In Vivo Studies of Receptors and Ion Channels with Unnatural Amino Acids. In: Köhrer, C., RajBhandary, U.L. (eds) Protein Engineering. Nucleic Acids and Molecular Biology, vol 22. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-540-70941-1_8

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