title: Site-directed alkylation of cysteine to test solvent accessibility of membrane proteins.
abstract: This protocol describes a detailed method to study the static and dynamic features of membrane proteins, as well as solvent accessibility, by utilizing the lactose permease of Escherichia coli (LacY) as a model. The method relies on the use of functional single-Cys mutants, an affinity tag and a PhosphoImager. The membrane-permeant, radioactive thiol reagent N-[ethyl-1-14C]ethylmaleimide ([14C]NEM) is used to detect site-directed alkylation of engineered single-Cys mutants in situ. The solvent accessibility of the Cys residues is also determined by blockage of [14C]NEM labeling with membrane-impermeant thiol reagents such as methanethiosulfonate ethylsulfonate (MTSES). The labeled proteins are purified by mini-scale affinity chromatography and analyzed by gel electrophoresis. Gels are dried and exposed to a PhosphoImager screen for 1-5 d, and incorporation of radioactivity is visualized. Initial results can be obtained in 24 h.
authors: Department of Physiology, University of California Los Angeles, Los Angeles, California, USA.
authors: Lan
authors: L
authors: Guan
authors: H Ronald
authors: HR
authors: Kaback
mesh_terms: D000345:Affinity Labels; D000478:Alkylation; D001671:Biochemistry / Q000379:methods; D002247:Carbon Isotopes; D002846:Chromatography, Affinity; D003545:Cysteine / Q000737:chemistry*; D004586:Electrophoresis; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D012997:Solvents; D013438:Sulfhydryl Compounds / Q000737:chemistry; D027981:Symporters
chemical_list: D000345:Affinity Labels; D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D012997:Solvents; D013438:Sulfhydryl Compounds; D027981:Symporters; D003545:Cysteine; D005033:Ethylmaleimide
references: Biochemistry. 2000 Sep 5;39(35):10656-61
references: 10978149
references: Biochemistry. 2000 Sep 5;39(35):10649-55
references: 10978148
references: Biochemistry. 2001 Sep 4;40(35):10491-9
references: 11523990
references: J Biol Chem. 2003 Mar 21;278(12):10641-8
references: 12471022
references: Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12148-52
references: 15304639
references: Methods Enzymol. 1974;31:698-709
references: 4609121
references: J Biol Chem. 1975 Jun 10;250(11):4291-6
references: 1092688
references: Science. 1992 Oct 9;258(5080):307-10
references: 1384130
references: Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6934-8
references: 8346199
references: Biochemistry. 1994 Jul 5;33(26):8074-81
references: 8025113
references: Biochemistry. 1996 Apr 2;35(13):3950-6
references: 8672426
references: Annu Rev Cell Dev Biol. 1997;13:457-512
references: 9442881
references: Methods Enzymol. 1998;293:123-45
references: 9711606
references: FASEB J. 1998 Oct;12(13):1281-99
references: 9761772
references: Biochemistry. 1998 Nov 10;37(45):15713-9
references: 9843376
references: Proc Natl Acad Sci U S A. 2005 Jun 14;102(24):8513-8
references: 15932938
references: Annu Rev Biophys Biomol Struct. 2006;35:67-91
references: 16689628
references: Proc Natl Acad Sci U S A. 2007 Jan 9;104(2):491-4
references: 17172438
references: J Biol Chem. 2007 Feb 16;282(7):4553-60
references: 17179147
references: Biochemistry. 2000 May 2;39(17):5097-103
references: 10819976
references: Nat Rev Mol Cell Biol. 2001 Aug;2(8):610-20
references: 11483994