Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography
Category
Published on
Type
journal-article
Author
Suraj Pandey and George Calvey and Andrea M. Katz and Tek Narsingh Malla and Faisal H. M. Koua and Jose M. Martin-Garcia and Ishwor Poudyal and Jay-How Yang and Mohammad Vakili and Oleksandr Yefanov and Kara A. Zielinski and Sasa Bajt and Salah Awel and Katarina Doerner and Matthias Frank and Luca Gelisio and Rebecca Jernigan and Henry Kirkwood and Marco Kloos and Jayanath Koliyadu and Valerio Mariani and Mitchell D. Miller and Grant Mills and Garrett Nelson and Jose L. Olmos and Alireza Sadri and Tokushi Sato and Alexandra Tolstikova and Weijun Xu and Abbas Ourmazd and John C. H. Spence and Peter Schwander and Anton Barty and Henry N. Chapman and Petra Fromme and Adrian P. Mancuso and George N. Phillips and Richard Bean and Lois Pollack and Marius Schmidt
Citation
Pandey, S. et al., 2021. Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography. IUCrJ, 8(6). Available at: http://dx.doi.org/10.1107/s2052252521008125.
Abstract
Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme–ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.
DOI
Funding
NSF-STC Biology with X-ray Lasers (NSF-1231306)