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

  • Nat Commun. 2021 Jul 22;12(1):4461. doi: 10.1038/s41467-021-24757-7.
Agata Butryn   #  1  2 ,  Philipp S Simon   #  3 ,  Pierre Aller   #  1  2 ,  Philip Hinchliffe  4 ,  Ramzi N Massad  3 ,  Gabriel Leen  5  6 ,  Catherine L Tooke  4 ,  Isabel Bogacz  3 ,  In-Sik Kim  3 ,  Asmit Bhowmick  3 ,  Aaron S Brewster  3 ,  Nicholas E Devenish  1 ,  Jürgen Brem  7 ,  Jos J A G Kamps  7  1 ,  Pauline A Lang  7 ,  Patrick Rabe  7 ,  Danny Axford  1 ,  John H Beale  1  8 ,  Bradley Davy  1  9 ,  Ali Ebrahim  1 ,  Julien Orlans  1  10 ,  Selina L S Storm  1  11 ,  Tiankun Zhou  1  2 ,  Shigeki Owada  12  13 ,  Rie Tanaka  12  14 ,  Kensuke Tono  12  13 ,  Gwyndaf Evans  1 ,  Robin L Owen  1 ,  Frances A Houle  15 ,  Nicholas K Sauter  3 ,  Christopher J Schofield  7 ,  James Spencer  4 ,  Vittal K Yachandra  3 ,  Junko Yano  3 ,  Jan F Kern  16 ,  Allen M Orville  17  18
Affiliations
  • 1. Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK.
  • 2. Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK.
  • 3. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
  • 4. School of Cellular and Molecular Medicine, University of Bristol, University Walk, Bristol, UK.
  • 5. PolyPico Technologies Ltd, Unit 10, Airways Technology Park, Rathmacullig West, Cork, Ireland.
  • 6. Department of Electronic and Computer Engineering, University of Limerick, Limerick, Ireland.
  • 7. Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, UK.
  • 8. Paul Scherrer Institut, Villigen PSI, Switzerland.
  • 9. School of Computing, University of Leeds, Leeds, UK.
  • 10. UMR0203, Biologie Fonctionnelle, Insectes et Interactions, Institut National des Sciences Appliquées de Lyon, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement, University of Lyon, Villeurbanne, France.
  • 11. European Molecular Biology Laboratory, Hamburg Outstation c/o DESY, Hamburg, Germany.
  • 12. RIKEN SPring-8 Center, Hyogo, Japan.
  • 13. Japan Synchrotron Radiation Research Institute, Hyogo, Japan.
  • 14. Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
  • 15. Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
  • 16. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. [email protected].
  • 17. Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK. [email protected].
  • 18. Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK. [email protected].
  • # Contributed equally.
PMID: 34294694 DOI: 10.1038/s41467-021-24757-7
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.

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