Time-resolved functional rotation crystallography probes protein dynamics during enzymatic catalysis

Abstract

Kinetic crystallography (KX) encompasses methods designed to characterize structural changes associated with protein dynamics and function. Here, we present Time-Resolved Functional Rotation Crystallography (TR-FRX), an integrated KX workflow combining room-temperature rotation crystallography on single crystals, nanolitre ligand delivery, and optional in crystallo optical spectroscopy using conventional synchrotron instrumentation. In TR-FRX, biochemical function is initiated by dispensing a nanolitre volume of ligand onto a single crystal while X-ray diffraction data are collected continuously. The resulting dataset is divided into sequential subdatasets, yielding a series of structural ensembles each averaged over the duration of their data collection time window. Together, these structural ensembles provide an overview of protein dynamics during ligand or substrate binding. We first applied TR-FRX to hen egg-white lysozyme in order to monitor N-acetylglucosamine binding and visualise associated local solvent reorganization. We then applied it to crystals of Chloroflexus aurantiacus malate dehydrogenase, first demonstrating by in crystallo spectroscopy that they are catalytically active, then revealing by X-ray diffraction ligand-associated solvent reorganization and active-site conformational changes over time windows ranging from hundreds of ms to tens of seconds. TR-FRX provides an accessible single-crystal workflow for establishing conditions in which ligand associated structural changes can be followed at room temperature, thereby generating preliminary data to guide future time-resolved serial crystallography experiments aimed at determining the structure of reaction intermediates.