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
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引用次数: 0
Abstract
A crystalline sample of CrLOV1 was optimized for serial crystallography. Time-resolved serial synchrotron crystallography provides high-resolution insights into structural changes of CrLOV1 from Δt = 2.5 ms up to Δt = 95 ms post-photoactivation, resolving the geometry of the thioether adduct and alteration of the C-terminal region implicated in the signal transduction.
Light–oxygen–voltage (LOV) domains are small photosensory flavoprotein modules that allow the conversion of external stimuli (sunlight) into intracellular signals responsible for various cell behaviors (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 result in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the activation cascade of the LOV domain to be observed 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 data collection, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the crystals obtained 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.
期刊介绍：
IUCrJ is a new fully open-access peer-reviewed journal from the International Union of Crystallography (IUCr).
The journal will publish high-profile articles on all aspects of the sciences and technologies supported by the IUCr via its commissions, including emerging fields where structural results underpin the science reported in the article. Our aim is to make IUCrJ the natural home for high-quality structural science results. Chemists, biologists, physicists and material scientists will be actively encouraged to report their structural studies in IUCrJ.
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