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.

本研究旨在捕捉细菌毒力因子AdhE所形成的大型多蛋白聚合物（spirosomes），在添加辅因子、底物以及本团队与其他合作者共同开发的抗毒力化合物时的毫秒级时间尺度动态响应。阐明AdhE——致病性大肠杆菌的关键代谢酶——的底物通道运输分子基础，以及抗毒力化合物对该螺旋体聚集体的影响机制，是理解这一动态响应的核心所在。时间分辨小角X射线散射（TR-SAXS）可在与生物及临床体系直接相关的溶液环境中，探究上述过程的发生时间尺度。本研究同时依托我们自主研发的新型X射线透明微流体停流芯片，以降低样品消耗量并最大化可获取的数据量。本研究将首次验证该芯片应用于临床相关蛋白质体系的能力。