Site-directed alkylation of cysteine to test solvent accessibility of membrane proteins.
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.
Guan
Lan
L
Department of Physiology, University of California Los Angeles, Los Angeles, California, USA.
Kaback
H Ronald
HR
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
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
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