An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography

Autor: Nicholas K. Sauter, Pauline A. Lang, Philipp S. Simon, Nicholas E. Devenish, Philip Hinchliffe, Vittal K. Yachandra, Junko Yano, Julien Orlans, Jürgen Brem, Gabriel Leen, Pierre Aller, Allen M. Orville, John H. Beale, James Spencer, Jos J. A. G. Kamps, A. Butryn, Catherine L. Tooke, Robin L. Owen, Ramzi N. Massad, Frances A. Houle, Kensuke Tono, Bradley Davy, Patrick Rabe, Jan Kern, Aaron S. Brewster, A. Ebrahim, Asmit Bhowmick, Tiankun Zhou, Gwyndaf Evans, Selina L. S. Storm, Shigeki Owada, Christopher J. Schofield, Isabel Bogacz, Danny Axford, Rie Tanaka, In-Sik Kim
Přispěvatelé: Biologie Fonctionnelle, Insectes et Interactions (BF2I), Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)
Jazyk: angličtina
Rok vydání: 2021
Předmět:
Zdroj: Nature Communications
Nature Communications, Nature Publishing Group, 2021, 12 (1), ⟨10.1038/s41467-021-24757-7⟩
Nature Communications, Vol 12, Iss 1, Pp 1-7 (2021)
Nature communications, vol 12, iss 1
Spencer, J, Hinchliffe, P, Tooke, C L, Kern, J F & Orville, A M 2021, ' An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography ', Nature Communications, vol. 12, no. 1, 4461 . https://doi.org/10.1038/s41467-021-24757-7
ISSN: 2041-1723
Popis: 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.
Currently many of the time resolved serial femtosecond (SFX) crystallography experiments are done with light driven protein systems, whereas the reaction initiation for non-light triggered enzymes remains a major bottle neck. Here, the authors present an expanded Drop-on-Tape system, where picoliter-sized droplets of a substrate or inhibitor are turbulently mixed with nanoliter sized droplets of microcrystal slurries, and they use it for time-resolved SFX measurements of inhibitor binding to lysozyme and secondly, binding of a β-lactam antibiotic to a bacterial serine β-lactamase.
Databáze: OpenAIRE
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