Abstract
Proteins are usually studied in well-defined buffer conditions, which differ substantially from those within a host cell. In some cases, the intracellular environment has an impact on the mechanism, which might be missed by in vitro experiments. IR difference spectroscopy previously has been applied to study the light-induced response of photoreceptors and photoenzymes in vitro Here, we established the in-cell IR difference (ICIRD) spectroscopy in the transmission and attenuated total reflection configuration to investigate the light-induced response of soluble proteins in living bacterial cells. ICIRD spectroscopy on the light, oxygen, or voltage (LOV) domains of the blue light receptors aureochrome and phototropin revealed a suppression of the response of specific secondary structure elements, indicating that the intracellular environment affects LOV photoreceptor mechanisms in general. Moreover, in-cell fluorescence spectroscopy disclosed that the intracellular environment slows down the recovery of the light-induced flavin adduct. Segment-resolved ICIRD spectroscopy on basic-region leucine zipper (bZIP)-LOV of aureochrome 1a from the diatom Phaeodactylum tricornutum indicated a signal progression from the LOV sensor to the bZIP effector independent of unfolding of the connecting A'α-helix, an observation that stood in contrast to in vitro results. This deviation was recapitulated in vitro by emulating the intracellular environment through the addition of the crowding agent BSA, but not by sucrose polymers. We conclude that ICIRD spectroscopy is a noninvasive, label-free approach for assessing conformational changes in receptors in living cells at ambient conditions. As demonstrated, these near-native responses may deviate from the mechanisms established under in vitro conditions.
Highlights
Photoreceptors are of central importance for many organisms, from bacteria, fungi, and plants to animals, for the determination of intensity and frequency of light in their environment and for the adequate response to beneficial or harmful conditions
We present here in-cell IR difference (ICIRD) spectroscopy in the transmission mode and the attenuated total reflection (ATR) configuration as an application for the investigation of the light-induced structural response of soluble photoreceptors in living cells
The LOV domain was moderately expressed in Escherichia coli BL21 using low-level induction
Summary
Photoreceptors are of central importance for many organisms, from bacteria, fungi, and plants to animals, for the determination of intensity and frequency of light in their environment and for the adequate response to beneficial or harmful conditions. The presence of small molecules, protein-protein interactions, the effect of macromolecular crowding, the different redox potential, and the different oxygen level might affect the light-induced response of a photoreceptor in cells It has been shown for plant cryptochrome 2 from Arabidopsis in intact insect cells that cellular conditions promote alternative reaction pathways [2]. To complement in vitro measurements, much effort has been put into establishing a broad field of techniques to study protein mechanisms in an intracellular environment [6] For soluble photoreceptors, such approaches include UV-visible spectroscopy on plant seedlings [7, 8] as well as fluorescence and EPR spectroscopy on insect cells [9, 10], which have been adapted to study the light-induced activation in situ. LOV receptors exhibit altered structural response in cells has been applied successfully in optogenetics as a light-dependent dimerizer for receptor tyrosine kinase activation [19, 20]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
