8QI9

CrPhotLOV1 dark state structure determined by serial synchrotron crystallography at room temperature

8QI9 の概要

• エントリーDOI: 10.2210/pdb8qi9/pdb
• 分子名称: Phototropin, FLAVIN MONONUCLEOTIDE (3 entities in total)
• 機能のキーワード: phototropin, flavin, electron transport, flavoprotein
• 由来する生物種: Chlamydomonas reinhardtii
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 15794.70

構造登録者

Gotthard, G.,Mous, S.,Weinert, T.,Maia, R.N.A.,James, D.,Dworkowski, F.,Gashi, D.,Antonia, F.,Wang, M.,Panepucci, E.,Ozerov, D.,Schertler, G.F.X.,Heberle, J.,Standfuss, J.,Nogly, P. (登録日: 2023-09-11, 公開日: 2024-07-24, 最終更新日: 2024-09-11)

主引用文献

Gotthard, G.,Mous, S.,Weinert, T.,Maia, R.N.A.,James, D.,Dworkowski, F.,Gashi, D.,Furrer, A.,Ozerov, D.,Panepucci, E.,Wang, M.,Schertler, G.F.X.,Heberle, J.,Standfuss, J.,Nogly, P.
Capturing the blue-light activated state of the Phot-LOV1 domain from Chlamydomonas reinhardtii using time-resolved serial synchrotron crystallography.
Iucrj, 11:792-808, 2024

PubMed Abstract: 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.

PubMed: 39037420
DOI: 10.1107/S2052252524005608

実験手法

X-RAY DIFFRACTION (1.87 Å)