학술논문
An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography.
Document Type
article
Author
Butryn, Agata; Simon, Philipp S; Aller, Pierre; Hinchliffe, Philip; Massad, Ramzi N; Leen, Gabriel; Tooke, Catherine L; Bogacz, Isabel; Kim, In-Sik; Bhowmick, Asmit; Brewster, Aaron S; Devenish, Nicholas E; Brem, Jürgen; Kamps, Jos JAG; Lang, Pauline A; Rabe, Patrick; Axford, Danny; Beale, John H; Davy, Bradley; Ebrahim, Ali; Orlans, Julien; Storm, Selina LS; Zhou, Tiankun; Owada, Shigeki; Tanaka, Rie; Tono, Kensuke; Evans, Gwyndaf; Owen, Robin L; Houle, Frances A; Sauter, Nicholas K; Schofield, Christopher J; Spencer, James; Yachandra, Vittal K; Yano, Junko; Kern, Jan F; Orville, Allen M
Source
Nature communications. 12(1)
Subject
Language
Abstract
Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.