Mix and Inject: Reaction Initiation by Diffusion for Time-Resolved Macromolecular Crystallography

TLDR: In this article, a time-resolved macromolecular crystallography (TRSMC) experiment is presented, in which a substrate is injected into the X-ray beam of a pulsed Xray source to start a reaction in an enzyme.

Abstract: Time-resolved macromolecular crystallography unifies structure determination with chemical kinetics, since the structures of transient states and chemical and kinetic mechanisms can be determined simultaneously from the same data. To start a reaction in an enzyme, typically, an initially inactive substrate present in the crystal is activated. This has particular disadvantages that are circumvented when active substrate is directly provided by diffusion. However, then it is prohibitive to use macroscopic crystals because diffusion times become too long. With small micro- and nanocrystals diffusion times are adequately short for most enzymes and the reaction can be swiftly initiated. We demonstrate here that a time-resolved crystallographic experiment becomes feasible by mixing substrate with enzyme nanocrystals which are subsequently injected into the X-ray beam of a pulsed X-ray source.