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.
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The authors acknowledge support from National Institutes of Health (NIH) grants DK51131 and DK06946, GM074929 and National Science Foundation (NSF) grant 0450970 (to H.R.K.).
The authors declare no competing financial interests.
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Guan, L., Ronald Kaback, H. Site-directed alkylation of cysteine to test solvent accessibility of membrane proteins. Nat Protoc 2, 2012–2017 (2007). https://doi.org/10.1038/nprot.2007.275
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