Guillaume Gotthard, Sandra Mous, Tobias Weinert, Raiza Nara Antonelli Maia, Daniel James, Florian Dworkowski, Dardan Gashi, Antonia Furrer, Dmitry Ozerov, Ezequiel Panepucci, Meitian Wang, Gebhard F. X. Schertler, Joachim Heberle, Joerg Standfuss, Przemyslaw Nogly: Capturing the blue-light activated state of the Phot-LOV1 domain from Chlamydomonas reinhardtii using time-resolved serial synchrotron crystallography. bioRxiv, 2023.

Abstract

Abstract: Light-Oxygen-Voltage (LOV) domains are small photosensory flavoprotein modules that allow converting external stimuli (sunlight) into intracellular signals responsible for various cell behavior (e.g., phototropism and chloroplast relocation). This ability relies on the light-induced formation of a covalent thioether adduct between a flavin chromophore and a reactive cysteine from the protein environment, which triggers a cascade of structural changes that results in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the observation of the activation cascade of the LOV domain in real-time, which has been elusive. In this study, we report a robust protocol for the production and stable delivery of microcrystals of the LOV domain of phototropin Phot-1 from Chlamydomonas reinhardtii (CrPhotLOV1) with a high-viscosity injector for time-resolved serial synchrotron crystallography (TR-SSX). The detailed process covers all aspects, from sample optimization to the actual data collection process, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the obtained crystals preserve the photoreactivity using infrared spectroscopy. Furthermore, the results of the TR-SSX experiment provide high-resolution insights into structural alterations of CrPhotLOV1 from Δt = 2.5 ms up to Δt = 95 ms post-photoactivation, including resolving the geometry of the thioether adduct and the C-terminal region implicated in the signal transduction process.

BibTeX (Download)

@unpublished{Gotthard2023,
title = {Capturing the blue-light activated state of the Phot-LOV1 domain from Chlamydomonas reinhardtii using time-resolved serial synchrotron crystallography},
author = {Guillaume Gotthard and Sandra Mous and Tobias Weinert and Raiza Nara Antonelli Maia and Daniel James and Florian Dworkowski and Dardan Gashi and Antonia Furrer and Dmitry Ozerov and Ezequiel Panepucci and Meitian Wang and Gebhard F. X. Schertler and Joachim Heberle and Joerg Standfuss and Przemyslaw Nogly},
url = {http://biorxiv.org/lookup/doi/10.1101/2023.11.06.565770},
doi = {10.1101/2023.11.06.565770},
year  = {2023},
date = {2023-11-06},
urldate = {2023-11-06},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract: Light-Oxygen-Voltage (LOV) domains are small photosensory flavoprotein modules that allow converting external stimuli (sunlight) into intracellular signals responsible for various cell behavior (e.g., phototropism and chloroplast relocation). This ability relies on the light-induced formation of a covalent thioether adduct between a flavin chromophore and a reactive cysteine from the protein environment, which triggers a cascade of structural changes that results in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the observation of the activation cascade of the LOV domain in real-time, which has been elusive. In this study, we report a robust protocol for the production and stable delivery of microcrystals of the LOV domain of phototropin Phot-1 from Chlamydomonas reinhardtii (CrPhotLOV1) with a high-viscosity injector for time-resolved serial synchrotron crystallography (TR-SSX). The detailed process covers all aspects, from sample optimization to the actual data collection process, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the obtained crystals preserve the photoreactivity using infrared spectroscopy. Furthermore, the results of the TR-SSX experiment provide high-resolution insights into structural alterations of CrPhotLOV1 from Δt = 2.5 ms up to Δt = 95 ms post-photoactivation, including resolving the geometry of the thioether adduct and the C-terminal region implicated in the signal transduction process.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}