Single-molecule photobleaching analysis is a useful approach for quantifying reactive membrane protein oligomerization in membranes. It provides a binary readout of a fluorophore attached to a protein subunit at dilute conditions. However, quantification of protein stoichiometry from this data requires information about the subunit labeling yields and whether there is non-specific background labeling. Any increases in subunit-specific labeling improves the ability to determine oligomeric states with confidence. A common strategy for site-specific labeling is by conjugation of a fluorophore bearing a thiol-reactive maleimide group to a substituted cysteine. Yet, cysteine reactivity can be difficult to predict as it depends on many factors such as solvent accessibility and electrostatics from the surrounding protein structure. Here we report a general methodology for screening potential cysteine labeling sites on purified membrane proteins. We present the results of two example systems for which the dimerization reactions in membranes has been characterized: (1) the CLC-ec1 Cl-/H+ antiporter, an Escherichia coli homologue of voltage-gated chloride ion channels in humans and (2) a mutant form of a member of the family of fluoride channels Fluc from Bordetella pertussis (Fluc-Bpe-N43S). To demonstrate how we identify such sites, we first discuss considerations of residue positions hypothesized to be suitable and describe the specific steps to rigorously assess site-specific labeling while maintaining the functional activity and robust single-molecule fluorescence signals. We find that our initial, well rationalized choices are not strong predictors of success, as rigorous testing of the labeling sites shows that only ≈30 % of sites end up being useful for single-molecule photobleaching studies.
Keywords: Cy5; Cysteine; Ellman; Maleimide; Membrane protein; Photobleaching.
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