Dynamic interactions among proteins of the bacterial flagellar type III secretion apparatus

Abstract

While interactions among proteins of the flagellar Type III secretion (T3S) export apparatus have been reported, few have been examined with respect to the dynamic relationships that must govern the mechanism of export. Using biolayer interferometry (BLI), an optical biosensing method, we have undertaken a survey of protein‐protein interactions among the soluble apparatus proteins (FlhAc, FlhBc, FliH, FliI, FliJ, FliK) and between apparatus proteins and export substrates. Proteins were purified prior to screening for binding partners. One of each potential pair was immobilized and then exposed to others in solution. Interactions identified by yes/no screens were subjected to rigorous characterization to measure affinity and rate constants. Our screen may uncover important interactions heretofore unknown because they are of insufficient affinity for copurification or affinity blotting experiments. Characterization will also allow us to pursue efforts such as identifying export signals in substrates by searching for kinetic changes in variant constructs. We are attempting to observe more‐than‐pairwise interactions. Kinetic characterization of dynamic interactions will allow us develop a better understanding of T3S. This work was supported by grants from the Public Health Service (R15GM080701), NSF (CHE 0922699), the Research Corporation (CC6900).