Room temperature crystallography and X-ray spectroscopy of metalloenzymes.

Autor:	Makita H; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States., Zhang M; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States., Yano J; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States. Electronic address: JYano@lbl.gov., Kern J; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States. Electronic address: JFKern@lbl.gov.
Jazyk:	angličtina
Zdroj:	Methods in enzymology [Methods Enzymol] 2023; Vol. 688, pp. 307-348. Date of Electronic Publication: 2023 Aug 16.
DOI:	10.1016/bs.mie.2023.07.009
Abstrakt:	The ultrashort (10s of femtoseconds) X-ray pulses generated by X-ray free electron lasers enable the measurement of X-ray diffraction and spectroscopic data from radiation-sensitive metalloenzymes at room temperature while mostly avoiding the effects of radiation damage usually encountered when performing such experiments at synchrotron sources. Here we discuss an approach to measure both X-ray emission and X-ray crystallographic data at the same time from the same sample volume. The droplet-on-tape setup described allows for efficient sample use and the integration of different reaction triggering options in order to conduct time-resolved studies with limited sample amounts. The approach is illustrated by two examples, photosystem II that catalyzes the light-driven oxidation of water to oxygen, and isopenicillin N synthase, an enzyme that catalyzes the double ring cyclization of a tripeptide precursor into the β-lactam isopenicillin and can be activated by oxygen exposure. We describe the necessary steps to obtain microcrystals of both proteins as well as the operation procedure for the drop-on-tape setup and details of the data acquisition and processing involved in this experiment. At the end, we present how the combination of time-resolved X-ray emission spectra and diffraction data can be used to improve the knowledge about the enzyme reaction mechanism. (Copyright © 2023. Published by Elsevier Inc.)
Databáze:	MEDLINE
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