Illuminating protein-protein interactions in the Escherichia coli chemotaxis signaling lattice through protein footprinting

The Escherichia coli chemotaxis signaling system has emerged as a paradigm for transmembrane signaling. Chemotactic signals are processed through a complex, interconnected lattice of signaling proteins. Characterizing the protein–protein interactions responsible for the sophisticated processing remains a problem at the frontier of chemotaxis and signal transduction. To study the protein interactions of the chemotaxis system, I employed cysteine (Cys) footprinting. The reactivity of Cys engineered in a protein of interest reports on the local chemical environment. Changes in Cys reactivity induced by protein binding can be mapped to define interaction surfaces. I have developed a footprinting strategy based on isotope-coded affinity tag (ICAT) reagents. The alkylation of Cys by the electrophilic ICAT reagents can be monitored using mass spectrometry. Because of their capacity for sample enrichment, we postulated that ICAT reagents would be ideal for footprinting in biologically-relevant milieu.;I showed that ICAT footprinting can be used to map a protein–protein interaction. Specifically, I examined the weak interaction (KD=17 μM) between the adaptor protein CheW and the kinase CheA. CheW mediates a functionally important bridging interaction between the transmembrane receptors and CheA. My footprinting studies reveal that binding regions can be mapped even in a low affinity complex. In addition, I showed that ICAT reagents can be used to footprint the interaction of CheW with the chemoreceptor Tsr even in the extremely high protein background of native membranes.;Another goal was to expand the utility of ICAT footprinting. Footprinting is complicated by the vast range of chemical reactivity conferred by a local protein environment. Surface residues react extremely rapidly; core residues react imperceptibly slowly. Rate changes can be difficult to detect at the extremes of reactivity. To address this issue, a series of ICAT reagents with a wide range of intrinsic alkylation rates was generated. Using this ICAT toolkit, we demonstrate footprinting of a deeply-buried protein core residue with a rapidly reacting ICAT reagent. We also use an ICAT reagent that reacts slowly to magnify the subtle footprint of a protein–protein interaction. Together, my studies highlight the utility of ICAT footprinting.