Capturing the blue-light activated state of the Phot-LOV1 domain fromChlamydomonas reinhardtiiusing time-resolved serial synchrotron crystallography

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 fromChlamydomonas 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 ofCrPhotLOV1 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.

A snapshot love story: what serial crystallography has done and will do for us

“…It could be shown that light-activation led to a change in the conformation of bound ATP, rendering it energetically favorable for enzyme-catalyzed conversion to cAMP (Chretien et al, 2024). The response of the FMN-containing Phot-LOV1 domain from Chlamydomonas reinhardtii to photoexcitation was investigated on slower time scales (2.5-92.5 ms) using timeresolved SSX (trSSX) at the Swiss Light Source (Gotthard et al, 2023).…”