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

被引:41
|
作者
Butryn, Agata [1 ,2 ]
Simon, Philipp S. [3 ]
Aller, Pierre [1 ,2 ]
Hinchliffe, Philip [4 ]
Massad, Ramzi N. [3 ]
Leen, Gabriel [5 ,6 ,7 ]
Tooke, Catherine L. [4 ]
Bogacz, Isabel [3 ]
Kim, In-Sik [3 ]
Bhowmick, Asmit [3 ]
Brewster, Aaron S. [3 ]
Devenish, Nicholas E. [1 ]
Brem, Jurgen [8 ]
Kamps, Jos J. A. G. [1 ,8 ]
Lang, Pauline A. [8 ]
Rabe, Patrick [8 ]
Axford, Danny [1 ]
Beale, John H. [1 ,14 ]
Davy, Bradley [1 ,15 ]
Ebrahim, Ali [1 ]
Orlans, Julien [1 ,9 ]
Storm, Selina L. S. [1 ]
Zhou, Tiankun [1 ,2 ]
Owada, Shigeki [10 ,11 ]
Tanaka, Rie [10 ,12 ]
Tono, Kensuke [10 ,11 ]
Evans, Gwyndaf [1 ]
Owen, Robin L. [1 ]
Houle, Frances A. [13 ]
Sauter, Nicholas K. [3 ]
Schofield, Christopher J. [8 ]
Spencer, James [5 ]
Yachandra, Vittal K. [3 ]
Yano, Junko [3 ]
Kern, Jan F. [3 ]
Orville, Allen M. [1 ,2 ]
机构
[1] Harwell Sci & Innovat Campus, Diamond Light Source, Didcot, Oxon, England
[2] Res Complex Harwell, Rutherford Appleton Lab, Didcot, Oxon, England
[3] Lawrence Berkeley Natl Lab, Mol Biophys & Integrated Bioimaging Div, Berkeley, CA 94720 USA
[4] Univ Bristol, Sch Cellular & Mol Med, Bristol, Avon, England
[5] PolyPico Technol Ltd, Unit 10,Airways Technol Pk, Cork, Ireland
[6] Univ Limerick, Dept Elect & Comp Engn, Limerick, Ireland
[7] Univ Oxford, Dept Chem, Chem Res Lab, Oxford, England
[8] Univ Lyon, Inst Natl Rech Agr Alimentat & Environm, Inst Natl Sci Appliquees Lyon, Biol Fonct Insectes & Interact,UMR0203, Villeurbanne, France
[9] RIKEN, SPring 8 Ctr, Tatsuno, Hyogo, Japan
[10] Japan Synchrotron Radiat Res Inst, Sayo, Hyogo, Japan
[11] Kyoto Univ, Grad Sch Med, Dept Cell Biol, Kyoto, Japan
[12] Lawrence Berkeley Natl Lab, Chem Sci Div, Berkeley, CA USA
[13] Paul Scherrer Inst, Villigen, Switzerland
[14] Univ Leeds, Sch Comp, Leeds, W Yorkshire, England
[15] DESY, European Mol Biol Lab, Hamburg Outstn, Hamburg, Germany
基金
英国惠康基金; 英国工程与自然科学研究理事会; 美国国家卫生研究院; 英国生物技术与生命科学研究理事会; “创新英国”项目;
关键词
SUBSTRATE-BINDING; PHOTOSYSTEM-II; LYSOZYME; SOFTWARE; DYNAMICS; EVOLUTION; MODEL;
D O I
10.1038/s41467-021-24757-7
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 beta-lactam antibiotic to a bacterial serine beta-lactamase.
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页数:7
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