title: Engineering the glutamate transporter homologue GltPh using protein semisynthesis.
abstract: Glutamate transporters catalyze the concentrative uptake of glutamate from synapses and are essential for normal synaptic function. Despite extensive investigations of glutamate transporters, the mechanisms underlying substrate recognition, ion selectivity, and the coupling of substrate and ion transport are not well-understood. Deciphering these mechanisms requires the ability to precisely engineer the transporter. In this study, we describe the semisynthesis of GltPh, an archaeal homologue of glutamate transporters. Semisynthesis allows the precise engineering of GltPh through the incorporation of unnatural amino acids and peptide backbone modifications. In the semisynthesis, the GltPh polypeptide is initially assembled from a recombinantly expressed thioester peptide and a chemically synthesized peptide using the native chemical ligation reaction followed by in vitro folding to the native state. We have developed a robust procedure for the in vitro folding of GltPh. Biochemical characterization of the semisynthetic GltPh indicates that it is similar to the native transporter. We used semisynthesis to substitute Arg397, a highly conserved residue in the substrate binding site, with the unnatural analogue, citrulline. Our studies demonstrate that Arg397 is required for high-affinity substrate binding, and on the basis of our results, we propose that Arg397 is involved in a Na+-dependent remodeling of the substrate binding site required for high-affinity Asp binding. We anticipate that the semisynthetic approach developed in this study will be extremely useful in investigating functional mechanisms in GltPh. Further, the approach developed in this study should also be applicable to other membrane transport proteins.
authors: Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health & Science University , 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239, United States.
authors: Paul J
authors: PJ
authors: Focke
authors: Alvin W
authors: AW
authors: Annen
authors: Francis I
authors: FI
authors: Valiyaveetil
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000138:chemical synthesis / Q000737:chemistry; D019843:Archaeal Proteins / Q000138:chemical synthesis / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
chemical_list: D027322:Amino Acid Transport System X-AG; D019843:Archaeal Proteins; D010455:Peptides
references: Nature. 1996 Oct 17;383(6601):634-7
references: 8857541
references: Chembiochem. 2014 Aug 18;15(12):1710-20
references: 24990307
references: Microbiol Mol Biol Rev. 1999 Jun;63(2):293-307
references: 10357852
references: Protein Expr Purif. 2005 May;41(1):207-34
references: 15915565
references: Nature. 2007 Jan 25;445(7126):387-93
references: 17230192
references: Nat Protoc. 2007;2(6):1528-35
references: 17571060
references: Neuron. 2000 Mar;25(3):695-706
references: 10774736
references: J Biol Chem. 2000 Dec 1;275(48):37436-42
references: 10978338
references: J Biol Chem. 2001 Apr 6;276(14):10775-81
references: 11148213
references: Prog Neurobiol. 2001 Sep;65(1):1-105
references: 11369436
references: J Am Chem Soc. 2002 Aug 7;124(31):9113-20
references: 12149015
references: Biochemistry. 2002 Sep 3;41(35):10771-7
references: 12196015
references: Annu Rev Biochem. 2003;72:249-89
references: 12626339
references: Nature. 2004 Oct 14;431(7010):811-8
references: 15483603
references: Annu Rev Biochem. 1988;57:957-89
references: 3052294
references: Int J Pept Protein Res. 1992 Sep-Oct;40(3-4):180-93
references: 1478777
references: Science. 1994 Nov 4;266(5186):776-9
references: 7973629
references: Nat Rev Neurosci. 2007 Dec;8(12):935-47
references: 17987031
references: J Biol Chem. 2009 Jun 26;284(26):17540-8
references: 19380583
references: Methods Enzymol. 2009;462:135-50
references: 19632473
references: Nature. 2009 Dec 17;462(7275):880-5
references: 19924125
references: Biochemistry. 2010 May 4;49(17):3511-3
references: 20349989
references: Annu Rev Biochem. 2010;79:413-44
references: 20307192
references: Neuropharmacology. 2011 Jan;60(1):172-81
references: 20708631
references: Biochemistry. 2011 Dec 6;50(48):10442-50
references: 22044112
references: Biochim Biophys Acta. 2012 Apr;1818(4):1055-66
references: 22100867
references: PLoS One. 2012;7(3):e33058
references: 22427946
references: Annu Rev Pharmacol Toxicol. 2013;53:211-29
references: 23294309
references: J Biol Chem. 2013 Mar 22;288(12):8250-7
references: 23393130
references: Nat Struct Mol Biol. 2013 May;20(5):634-40
references: 23563139
references: Nat Struct Mol Biol. 2013 Oct;20(10):1224-6
references: 24013209
references: Physiol Rev. 2013 Oct;93(4):1621-57
references: 24137018
references: J Biol Chem. 2013 Dec 6;288(49):35266-76
references: 24155238
references: Pflugers Arch. 2014 Jan;466(1):3-24
references: 24240778
references: Elife. 2014;3:e02283
references: 24842876
references: J Neurosci. 1998 Dec 1;18(23):9620-8
references: 9822723