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.

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Figure 1: Diagram for the application of N-ethylmaleimide (NEM) labeling.
Figure 2: Diagram for the strategy of testing solvent accessibility of Cys.
Figure 3
Figure 4: Accessibility of single-Cys LacY mutants to methanethiosulfonate ethylsulfonate (MTSES) and effect of ligand.

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Acknowledgements

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.).

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Correspondence to H Ronald Kaback.

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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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