Abstract
The topology of membrane proteins and enzymes can be determined using various methods including reporter protein fusions and accessibility of cysteine residues to alkylating agents. Here we describe a variation of the substituted cysteine accessibility method to determine membrane topology and activity of enzymes containing an active site cysteine. Membrane topology of proteins can be predicted using different programs and the actual membrane topology can be determined by monitoring the accessibility of cysteine residues introduced in periplasmic (exposed) or cytoplasmic (not exposed) loops to alkylating agents. A two-step protocol is described where whole Escherichia coli (E. coli) cells are first treated with or without a membrane impermeable thiol reagent (2-sulfonatoethyl)-methane thiosulfonate (MTSES) and subsequently labeled with an alkylating reagent maleimide polyethyleneglycol (malPEG). When cysteine residues are accessible to MTSES, and thus exposed to (or accessible from) the periplasm, their free thiol groups covalently react with MTSES and consequently, are blocked for alkylation with malPEG. The thiol groups of cytoplasmic or membrane-embedded cysteine residues are not accessible to MTSES and proteins can be alkylated with malPEG resulting in an increase in molecular weight of 5 kDa. In the second part of the protocol, accessibility of cysteine residues is used to address the acylation state of enzymes that form stable thioester acyl intermediates. Thioesters can be specifically cleaved by neutral hydroxylamine, leading to a free thiol group of the active site cysteine that can then be alkylated with malPEG.
Materials and Reagents
Equipment
Procedure
I. Accessibility of cysteine residues using thiol specific and alkylating reagents
II. Accessibility of cysteine to alkylating agents after cleavage of thioester bond Follow protocol described in A.
Representative data
Figure 1. Example of malPEG alkylation profile of Apolipoprotein N-acyltransferase (Lnt) of E. coli. Cysteine residues 23 and 62 were substituted by alanine, leaving a single active site cysteine residue C387. Lnt forms a thioester acyl-enzyme intermediate in vivo with C387 and is blocked for reaction with malPEG. Upon treatment of cells with hydroxylamine (HA), the thioester bond is cleaved, leaving a free thiol group of C387 and alkylation by malPEG. Please see Gélis-Jeanvoine et al. (2015) for representative examples.
Notes
Results may vary depending on the membrane protein studied. It is highly recommended that negative and positive controls are included to validate technical details of the procedure. For example, use of empty vector, a cysteine-less variant of the protein of interest and inactive mutants with substituted cysteine residues are recommended.
Recipes
Acknowledgments
This work was financed by the Institut Pasteur.
References
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