Ligand Binding: recording events in the active and alternative binding sites using time-resolved crystallography

On a molecular level, biological function is closely linked to the dynamic processes of enzymes. However, capturing these conformational changes, especially during catalysis, has proven difficult using conventional experimental methods. Uniquely, time-resolved X-ray crystallography (TRX) can provide insight into these dynamic changes, but is often compounded with substantial experimental challenges. To surmount these challenges and enable TRX on typical beamlines, we have pioneered cryo-trapping TRX. Our SPITROBOT crystal plunger enables to move beyond canonical static imaging of proteins and instead capture short-lived reaction intermediates, that report on dynamic biochemical processes as they unfold. Here we aim to demonstrate that the next generation plunger ‘SPITROBOT-2’ enables reaction quenching within 25 ms and allows following the dynamic events during ligand binding and catalysis for several model systems.

从分子层面来看，生物功能与酶的动态过程紧密关联。然而，借助常规实验手段捕捉这些构象变化——尤其是催化过程中的构象变化——已被证实极具挑战。唯有时间分辨X射线晶体学（time-resolved X-ray crystallography, TRX）能够深入解析这类动态变化，但该方法往往伴随大量棘手的实验难题。为克服上述难题并在常规光束线站上开展TRX实验，我们率先开发了低温捕获TRX技术。我们研发的SPITROBOT晶体推杆装置，打破了蛋白质静态成像的常规范式，能够捕捉短寿命反应中间体，而这些中间体可实时反映动态生化过程的发生与演进。本研究旨在验证新一代SPITROBOT-2推杆装置可在25毫秒内完成反应淬灭，并能够追踪多个模型体系中配体结合与催化过程中的动态事件。