Title:
Isolated bacterial chemosensory array possesses quasi- and ultrastable components: functional links between array stability, cooperativity, and order.

Abstract:
Bacteria utilize a large multiprotein chemosensory array to sense attractants and repellents in their environment. The array is a hexagonal lattice formed from three core proteins: a transmembrane receptor, the His kinase CheA, and the adaptor protein CheW. The resulting, highly networked array architecture yields several advantages including strong positive cooperativity in the attractant response and rapid signal transduction through the preformed, integrated signaling circuit. Moreover, when isolated from cells or reconstituted in isolated bacterial membranes, the array possesses extreme kinetic stability termed ultrastability (Erbse and Falke (2009) Biochemistry 48:6975-87) and is the most long-lived multiprotein enzyme complex described to date. The isolated array retains kinase activity, attractant regulation, and its bound core proteins for days or more at 22 °C. The present work quantitates this ultrastability and investigates its origin. The results demonstrate that arrays consist of two major components: (i) a quasi-stable component with a lifetime of 1-2 days that decays due to slow proteolysis of CheA kinase in the lattice and (ii) a truly ultrastable component with a lifetime of ~20 days that is substantially more protected from proteolysis. Following proteolysis of the quasi-stable component the apparent positive cooperativity of the array increases, arguing the quasi-stable component is not as cooperative as the ultrastable component. Introduction of structural defects into the array by coupling a bulky probe to a subset of receptors reveals that modification of only 2% of the receptor population is sufficient to abolish ultrastability, supporting the hypothesis that the ultrastable component requires a high level of array spatial order. Overall, the findings are consistent with a model in which the quasi- and ultrastable components arise from distinct regions of the array, such that the ultrastable regions possess more extensive, better-ordered, multivalent interconnectivities between core components, thereby yielding extraordinary stability and cooperativity. Furthermore, the findings indicate that the chemosensory array is a promising platform for the development of ultrastable biosensors.