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

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.

Authors:
ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [1];  [3];  [2];  [4];  [3];  [2];  [2];  [2]; ORCiD logo [2];  [5]; ORCiD logo [6]; ORCiD logo [7]; ORCiD logo [6];  [6]; ORCiD logo [5];  [8];  [9];  [5] more »; ORCiD logo [10]; ORCiD logo [11];  [1];  [12];  [13];  [12]; ORCiD logo [5]; ORCiD logo [5];  [2]; ORCiD logo [2]; ORCiD logo [6]; ORCiD logo [3];  [2]; ORCiD logo [2]; ORCiD logo [2]; ORCiD logo [1] « less
+ Show Author Affiliations
  1. Harwell Science and Innovation Campus, Didcot (United Kingdom); Research Complex at Harwell, Didcot (United Kingdom)
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  3. Univ. of Walk, Bristol (United Kingdom)
  4. PolyPico Technologies Ltd., Cork (Ireland); Univ. of Limerick (Ireland)
  5. Harwell Science and Innovation Campus, Didcot (United Kingdom)
  6. Univ. of Oxford (United Kingdom)
  7. Harwell Science and Innovation Campus, Didcot (United Kingdom); Univ. of Oxford (United Kingdom)
  8. Paul Scherrer Inst. (PSI), Villigen (Switzerland); Harwell Science and Innovation Campus, Didcot (United Kingdom)
  9. Univ. of Leeds, Leeds (United Kingdom); Harwell Science and Innovation Campus, Didcot (United Kingdom)
  10. Harwell Science and Innovation Campus, Didcot (United Kingdom); Univ. of Lyons, Villeurbanne (France)
  11. European Molecular Biology Lab. (EMBL), Hamburg (Germany); Harwell Science and Innovation Campus, Didcot (United Kingdom)
  12. Inst. of Physical and Chemical Research (RIKEN), Hyogo (Japan); Japan Synchrotron Radiation Research Institute, Sayo, Hyogo (Japan)
  13. Inst. of Physical and Chemical Research (RIKEN), Hyogo (Japan); Kyoto Univ. (Japan)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division; National Institutes of Health (NIH); Biotechnology and Biological Sciences Research Council; Wellcome Investigator Award in Science; Royal Society Wolfson Fellowship; AMED
OSTI Identifier:
1825694
Grant/Contract Number:  
AC02-05CH11231; GM117126; GM55302; GM110501; GM126289; GM133081; 102593; 210734/Z/18/Z; RSWF/R2/182017; BB/J014400/1; EP/L015838/1; JP20am0101070
Resource Type:
Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 12; Journal Issue: 1; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; computational models; optical spectroscopy; structural biology; x-ray crystallography

Citation Formats

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 J.A.G., Lang, Pauline A., Rabe, Patrick, Axford, Danny, Beale, John H., Davy, Bradley, Ebrahim, Ali, Orlans, Julien, Storm, Selina L. S., 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., and Orville, Allen M. An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography. United States: N. p., 2021. Web. doi:10.1038/s41467-021-24757-7.
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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 J.A.G., Lang, Pauline A., Rabe, Patrick, Axford, Danny, Beale, John H., Davy, Bradley, Ebrahim, Ali, Orlans, Julien, Storm, Selina L. S., 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. An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography. United States. https://doi.org/10.1038/s41467-021-24757-7
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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 J.A.G., Lang, Pauline A., Rabe, Patrick, Axford, Danny, Beale, John H., Davy, Bradley, Ebrahim, Ali, Orlans, Julien, Storm, Selina L. S., 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., and Orville, Allen M. Thu . "An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography". United States. https://doi.org/10.1038/s41467-021-24757-7. https://www.osti.gov/servlets/purl/1825694.
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@article{osti_1825694,
title = {An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography},
author = {Butryn, Agata and Simon, Philipp S. and Aller, Pierre and Hinchliffe, Philip and Massad, Ramzi N. and Leen, Gabriel and Tooke, Catherine L. and Bogacz, Isabel and Kim, In-Sik and Bhowmick, Asmit and Brewster, Aaron S. and Devenish, Nicholas E. and Brem, Jürgen and Kamps, Jos J.A.G. and Lang, Pauline A. and Rabe, Patrick and Axford, Danny and Beale, John H. and Davy, Bradley and Ebrahim, Ali and Orlans, Julien and Storm, Selina L. S. and Zhou, Tiankun and Owada, Shigeki and Tanaka, Rie and Tono, Kensuke and Evans, Gwyndaf and Owen, Robin L. and Houle, Frances A. and Sauter, Nicholas K. and Schofield, Christopher J. and Spencer, James and Yachandra, Vittal K. and Yano, Junko and Kern, Jan F. and Orville, Allen M.},
abstractNote = {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.},
doi = {10.1038/s41467-021-24757-7},
journal = {Nature Communications},
number = 1,
volume = 12,
place = {United States},
year = {Thu Jul 22 00:00:00 EDT 2021},
month = {Thu Jul 22 00:00:00 EDT 2021}
}
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