The instability of some proteins can hamper in vitro studies. This is true for the membrane-bound aerotaxis receptor, Aer, which exhibits significant proteolysis during the preparation of membrane vesicles. Permeabilized cells can closely mimic in vivo conditions, maintaining the intracellular milieu and geometry of interacting domains. Here, we describe an optimized method for determining solvent accessibility in permeabilized Escherichia coli cells. In this method, E. coli expressing Aer with a series of cysteine replacements are treated with toluene and ethanol, after which a large sulfhydryl reactive probe, PEG-mal, is added. PEGylated protein is separated from un-PEGylated protein by its apparent size difference on SDS-PAGE. The extent to which each cysteine residue becomes PEGylated is then used as a measure of solvent accessibility. When a library of single-Cys replacements is mapped, regions of low accessibility can suggest interacting protein surfaces. We successfully used this method to reveal inaccessible surfaces on both the Aer PAS and HAMP domains that were then shown by disulfide cross-linking to interact.
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We thank Claudia A. Studdert at the Universidad Nacional de Mar del Plata in Argentina for personally communicating her unpublished PEGylation protocol. The development and communication of the optimized method and the work described here was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers R01GM029481 to Barry L. Taylor, and R01GM108655 to Kylie J. Watts, respectively.
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