Disulfide Trapping and Spectroscopic Studies of Bacterial Chemosensory Core Signaling Complexes: Probing Molecular Mechanisms of Complex Assembly and Receptor-Regulated On-Off Switching

Abstract

The core unit of the bacterial chemosensory array is multi-protein complex comprised of 6 transmembrane chemoreceptor homodimers, 1 CheA His kinase homodimer, and 2 CheW adaptor protein monomers. We are reconstituting core units on isolated bacterial membranes, yielding individual core units and oligomers of core units ranging in size up to small hexagonal arrays. This approach generates functional, membrane-bound core complexes and allows incorporation of modified kinase and adaptor proteins possessing pairs of engineered Cys residues for disulfide trapping, or spectroscopic probes for fluorescence or EPR studies. The resulting core complexes display native receptor-stimulated kinase activities that are fully regulated by attractant binding to the receptor, or covalent modification of the receptor adaptation sites. The findings shed new light on the kinetics and order of core complex assembly, and provide insights into the molecular mechanisms underlying receptor-mediated kinase on-off switching.