title: Guided reconstitution of membrane protein fragments.
abstract: Structural analysis by NMR of G protein-coupled receptors (GPCRs) has proven to be extremely challenging. To reduce the number of peaks in the NMR spectra by segmentally labeling a GPCR, we have developed a Guided Reconstitution method that includes the use of charged residues and Cys activation to drive heterodimeric disulfide bond formation. Three different cysteine-activating reagents: 5-5'-dithiobis(2-nitrobenzoic acid) [DTNB], 2,2'-dithiobis(5-nitropyridine) [DTNP], and 4,4'-dipyridyl disulfide [4-PDS] were analyzed to determine their efficiency in heterodimer formation at different pHs. Short peptides representing the N-terminal (NT) and C-terminal (CT) regions of the first extracellular loop (EL1) of Ste2p, the Saccharomyces cerevisiae alpha-factor mating receptor, were activated using these reagents and the efficiencies of activation and rates of heterodimerization were analyzed. Activation of NT peptides with DTNP and 4-PDS resulted in about 60% yield, but heterodimerization was rapid and nearly quantitative. Double transmembrane domain protein fragments were biosynthesized and used in Guided Reconstitution reactions. A 102-residue fragment, 2TM-tail [Ste2p(G31-I120C)], was heterodimerized with CT-EL1-tail(DTNP) at pH 4.6 with a yield of ∼75%. A 132-residue fragment, 2TMlong-tail [Ste2p(M1-I120C)], was expressed in both unlabeled and (15)N-labeled forms and used with a peptide comprising the third transmembrane domain, to generate a 180-residue segmentally labeled 3TM protein that was found to be segmentally labeled using [(15)N,(1)H]-HSQC analysis. Our data indicate that the Guided Reconstitution method would be applicable to the segmental labeling of a membrane protein with 3 transmembrane domains and may prove useful in the preparation of an intact reconstituted GPCR for use in biophysical analysis and structure determination.
authors.affiliation: Department of Chemistry, The College of Staten Island, City University of New York (CUNY), Staten Island, NY, 10314.
authors.forename: Leah S
authors.initials: LS
authors.lastname: Cohen
authors.forename: Boris
authors.initials: B
authors.lastname: Arshava
authors.forename: Sarah
authors.initials: S
authors.lastname: Kauffman
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Mathew
authors.forename: Katrina E
authors.initials: KE
authors.lastname: Fracchiolla
authors.forename: Fa-Xiang
authors.initials: FX
authors.lastname: Ding
authors.forename: Mark E
authors.initials: ME
authors.lastname: Dumont
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Becker
authors.forename: Fred
authors.initials: F
authors.lastname: Naider
mesh_terms: D000595:Amino Acid Sequence; D001671:Biochemistry / Q000379:methods*; D003488:Cyanogen Bromide / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000737:chemistry; D055503:Protein Multimerization; D051964:Receptors, Mating Factor / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D013997:Time Factors
chemical_list: D004220:Disulfides; D008565:Membrane Proteins; D010455:Peptides; D051964:Receptors, Mating Factor; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D003545:Cysteine; D003488:Cyanogen Bromide
keywords: GPCR; guided reconstitution; heterodisulfide formation; membrane protein fragments; segmental-labeling
references.citation: J Am Chem Soc. 2002 Aug 7;124(31):9113-20
references.pmid: 12149015
references.citation: Methods. 2007 Apr;41(4):370-80
references.pmid: 17367709
references.citation: Chem Commun (Camb). 2004 Aug 7;(15):1722-3
references.pmid: 15278155
references.citation: Biophys J. 2009 Apr 22;96(8):3187-96
references.pmid: 19383463
references.citation: J Biol Chem. 2010 Dec 10;285(50):39425-36
references.pmid: 20923758
references.citation: Angew Chem Int Ed Engl. 2011 Apr 18;50(17):3988-92
references.pmid: 21433227
references.citation: Biochim Biophys Acta. 2013 Feb;1828(2):776-84
references.pmid: 23159810
references.citation: Acc Chem Res. 2009 Jan 20;42(1):107-16
references.pmid: 18939858
references.citation: Trends Pharmacol Sci. 2012 May;33(5):249-60
references.pmid: 22465153
references.citation: Biochemistry. 2001 Sep 18;40(37):11106-13
references.pmid: 11551208
references.citation: J Am Chem Soc. 2010 Jul 21;132(28):9526-7
references.pmid: 20405940
references.citation: Pharmacol Rev. 2011 Dec;63(4):901-37
references.pmid: 21969326
references.citation: J Biol Chem. 2007 Apr 6;282(14):10387-97
references.pmid: 17293349
references.citation: Methods Enzymol. 2009;462:151-75
references.pmid: 19632474
references.citation: Chem Soc Rev. 2009 Feb;38(2):338-51
references.pmid: 19169452
references.citation: Proc Natl Acad Sci U S A. 2010 Dec 28;107(52):22481-6
references.pmid: 21156831
references.citation: J Am Chem Soc. 2007 Mar 7;129(9):2432-3
references.pmid: 17284035
references.citation: J Biol Chem. 1995 Jul 28;270(30):18000-6
references.pmid: 7629108
references.citation: Annu Rev Med. 2004;55:27-39
references.pmid: 14746508
references.citation: J Pept Sci. 2002 Apr;8(4):172-80
references.pmid: 11991206
references.citation: Methods Mol Biol. 2004;237:105-20
references.pmid: 14501043
references.citation: Trends Pharmacol Sci. 2012 Jan;33(1):17-27
references.pmid: 22032986
references.citation: Biochim Biophys Acta. 2011 Nov;1808(11):2674-84
references.pmid: 21791199
references.citation: AAPS J. 2011 Mar;13(1):92-8
references.pmid: 21234731
references.citation: Drug Discov Today. 2009 Dec;14(23-24):1130-5
references.pmid: 19733256
references.citation: Biopolymers. 2012;98(5):485-500
references.pmid: 23203693
references.citation: Biopolymers. 2006 Sep;83(1):46-61
references.pmid: 16634087
references.citation: Methods Enzymol. 1991;194:3-21
references.pmid: 2005794
references.citation: Biochemistry. 1999 Jan 12;38(2):682-95
references.pmid: 9888809
references.citation: J Struct Funct Genomics. 2006 Mar;7(1):51-64
references.pmid: 16850177
references.citation: BMC Biol. 2009 Aug 13;7:50
references.pmid: 19678920
references.citation: J Pept Sci. 2010 May;16(5):213-8
references.pmid: 20401922
references.citation: Science. 1988 Jun 3;240(4857):1310-6
references.pmid: 2836950
references.citation: Biochem Biophys Res Commun. 2009 Dec 18;390(3):441-6
references.pmid: 19766594
references.citation: Biochemistry. 2008 Mar 25;47(12):3705-14
references.pmid: 18302338
references.citation: J Biol Chem. 1996 Mar 29;271(13):7860-7
references.pmid: 8631831
references.citation: Biopolymers. 2008;90(2):117-30
references.pmid: 18260136
references.citation: J Biomol NMR. 2008 Dec;42(4):257-69
references.pmid: 18937032
references.citation: Trends Pharmacol Sci. 2012 May;33(5):268-72
references.pmid: 22503476
references.citation: Anal Biochem. 2004 Dec 1;335(1):168-70
references.pmid: 15519585
references.citation: Nat Rev Drug Discov. 2006 Dec;5(12):993-6
references.pmid: 17139284
references.citation: Biochemistry. 2010 Jun 22;49(24):5007-15
references.pmid: 20420459
references.citation: J Am Chem Soc. 2012 Jul 18;134(28):11338-41
references.pmid: 22734434
references.citation: Nature. 2012 Nov 29;491(7426):779-83
references.pmid: 23086146
references.citation: Angew Chem Int Ed Engl. 2004 May 3;43(19):2504-7
references.pmid: 15127436
references.citation: PLoS One. 2012;7(10):e46290
references.pmid: 23056277