Millisecond kinetics of AdhE spirosome conformational response to cofactors, substrates and anti-virulence drug candidates

We aim to capture the millisecond timescale response of large, multi-protein polymers (spirosomes) of the bacterial virulence factor AdhE in response to the addition of cofactors and substrates, and anti-virulence compounds being developed by us in collaboration with others. Understanding this response is key to discerning the molecular basis of substrate chanelling by AdhE, a key metabolic enzyme of pathogenic E. coli, and how the spirosomes are impacted by the anti-virulence compounds. TR-SAXS has the capability to explore the timeframe on which these processes occur and in the solution environment directly relevant to the biological and clinical system. This work also relies on the use of a novel X-ray transparent microfluidic stopped-flow chip we have developed in-house to minimise sample volumes and maximise the number of data we can generate. This work will constitute the first demonstration of the capability of this chip for a clinically relevant protein system.