@inbook{6351e8a7350f4644932aec0808094b6f,
title = "Analyzing Protein Domain Interactions in Chemoreceptors by In Vivo PEGylation.",
abstract = "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.",
keywords = "Aer, Aerotaxis, Chemoreceptor, Disulfide cross-linking, HAMP domain, PAS domain, PEG-maleimide, Solvent accessibility, Toluene{\textendash}ethanol, Intercellular Signaling Peptides and Proteins, Cell Membrane Permeability, Models, Molecular, Solvents/chemistry, Chemotaxis, Carrier Proteins/chemistry, Escherichia coli/physiology, Cell Membrane/metabolism, Escherichia coli Proteins/chemistry, Polyethylene Glycols/chemistry, Serine/chemistry, Protein Conformation, Protein Interaction Domains and Motifs, Mutation",
author = "Watts, \{Kylie J.\} and Johnson, \{Mark S.\}",
note = "Publisher Copyright: {\textcopyright} 2018, Springer Science+Business Media, LLC.",
year = "2018",
month = feb,
day = "11",
doi = "10.1007/978-1-4939-7577-8\_13",
language = "American English",
isbn = "978-1-4939-7576-1",
series = "Methods in molecular biology (Clifton, N.J.)",
publisher = "Humana Press Inc.",
pages = "137--145",
booktitle = "Bacterial Chemosensing",
}