title: Measuring transmembrane helix interaction strengths in lipid bilayers using steric trapping.
abstract: We have developed a method to measure strong transmembrane (TM) helix interaction affinities in lipid bilayers that are difficult to measure by traditional dilution methods. The method, called steric trapping, couples dissociation of biotinylated TM helices to a competitive binding by monovalent streptavidin (mSA), so that dissociation is driven by the affinity of mSA for biotin and mSA concentration. By adjusting the binding affinity of mSA through mutation, the method can obtain dissociation constants of TM helix dimers (K d,dimer) over a range of six orders of magnitudes. The K d,dimer limit of measurable target interaction is extended 3-4 orders of magnitude lower than possible by dilution methods. Thus, steric trapping opens up new opportunities to study the folding and assembly of α-helical membrane proteins in lipid bilayer environments. Here we provide detailed methods for applying steric trapping to a TM helix dimer.
authors: Department of Chemistry, Michigan State University, East Lansing, MI, USA.
authors: Heedeok
authors: H
authors: Hong
authors: Yu-Chu
authors: YC
authors: Chang
authors: James U
authors: JU
authors: Bowie
mesh_terms: D000595:Amino Acid Sequence; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000378:metabolism; D011721:Pyrenes / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D008051:Lipid Bilayers; D008565:Membrane Proteins; D011510:Proteolipids; D011721:Pyrenes; D013438:Sulfhydryl Compounds; C020485:proteoliposomes; D019809:Streptavidin; C030984:pyrene
references: J Mol Biol. 1997 Sep 19;272(2):266-75
references: 9299353
references: Cell Mol Life Sci. 2006 Dec;63(24):2992-3017
references: 17086379
references: J Biol Chem. 1992 Apr 15;267(11):7683-9
references: 1560003
references: Protein Sci. 2000 Jun;9(6):1246-53
references: 10892817
references: J Am Chem Soc. 2011 Jul 27;133(29):11389-98
references: 21682279
references: Structure. 2008 Jul;16(7):991-1001
references: 18611372
references: Anal Biochem. 2005 May 1;340(1):154-64
references: 15802141
references: Annu Rev Biophys Biomol Struct. 1999;28:319-65
references: 10410805
references: J Am Chem Soc. 2010 Mar 17;132(10):3628-35
references: 20158179
references: Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14340-4
references: 11724930
references: Biophys J. 2003 Nov;85(5):3097-105
references: 14581210
references: Methods Enzymol. 2009;455:213-36
references: 19289208
references: Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1754-8
references: 7878054
references: Nat Methods. 2006 Apr;3(4):267-73
references: 16554831
references: Protein Sci. 2000 May;9(5):878-85
references: 10850797
references: J Mol Biol. 2002 Oct 25;323(3):563-71
references: 12381309
references: J Biol Chem. 1960 Mar;235:769-75
references: 13793161
references: J Mol Biol. 2004 Aug 20;341(4):991-8
references: 15289100
references: Proc Natl Acad Sci U S A. 2010 Nov 16;107(46):19802-7
references: 21041662
references: Curr Opin Struct Biol. 2008 Aug;18(4):412-9
references: 18539023
references: Anal Biochem. 2004 Aug 15;331(2):340-8
references: 15265740
references: Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):14772-7
references: 14657351
references: J Am Chem Soc. 2009 Oct 7;131(39):13914-5
references: 19739627
references: Protein Sci. 1999 Apr;8(4):921-9
references: 10211839
references: J Mol Biol. 1999 Oct 29;293(3):639-51
references: 10543956
references: Curr Opin Struct Biol. 2011 Aug;21(4):460-6
references: 21763125