An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
22 07 2021
22 07 2021
Historique:
received:
04
02
2021
accepted:
01
07
2021
entrez:
23
7
2021
pubmed:
24
7
2021
medline:
4
8
2021
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 34294694
doi: 10.1038/s41467-021-24757-7
pii: 10.1038/s41467-021-24757-7
pmc: PMC8298390
doi:
Substances chimiques
Avian Proteins
0
Bacterial Proteins
0
Enzymes
0
Recombinant Proteins
0
hen egg lysozyme
EC 3.2.1.-
Muramidase
EC 3.2.1.17
beta-lactamase CTX-M-15
EC 3.5.2.-
beta-Lactamases
EC 3.5.2.6
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
4461Subventions
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/J014400/1
Pays : United Kingdom
Organisme : NIGMS NIH HHS
ID : R01 GM126289
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM110501
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM055302
Pays : United States
Organisme : Wellcome Trust
ID : 210734/Z/18/Z
Pays : United Kingdom
Organisme : NIGMS NIH HHS
ID : R56 GM055302
Pays : United States
Organisme : Biotechnology and Biological Sciences Research Council
ID : 102593
Pays : United Kingdom
Organisme : NIGMS NIH HHS
ID : F32 GM133081
Pays : United States
Organisme : Wellcome Trust
Pays : United Kingdom
Organisme : NIGMS NIH HHS
ID : R01 GM117126
Pays : United States
Informations de copyright
© 2021. Crown.
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