Time-resolved infrared difference spectroscopy in cells: Response of the basic region leucine zipper of aureochrome

Abstract

Aureochromes are light, oxygen, voltage (LOV) proteins and central blue-light receptors in algae acting as light-gated transcription factors. The C-terminal LOV domain mediates blue-light recognition and the basic region leucine zipper (bZIP) domain binds a specific DNA motif as effector. LOV domains from aureochromes have been successfully applied in optogenetic tools. The light-induced response of aureochromes has been studied by a variety of biophysical techniques, but the mechanism of signal progression from LOV to bZIP remains unclear. We studied the bZIP-LOV module of aureochrome1a from the diatom Phaeodactylum tricornutum using time-resolved rapid-scan FTIR difference spectroscopy. Time-resolved difference spectra of bZIP-LOV in vitro revealed a time constant of 5 s for the formation of a light state dimer of the LOV domains and the concomitant loss of α-helical elements in the bZIP domain. To verify these observations in a near-native environment, in-cell infrared difference spectroscopy (ICIRD) was extended from a steady state to a time-resolved technique using LOV domains in bacterial cells. We established a time-resolved in-cell method with a resolution of 7.6 ms after the laser pulse. Using this technique, the response of bZIP-LOV was followed in living bacterial cells and the light-induced partial unfolding of bZIP was confirmed to take place in cells in a similar time range as in vitro. These results provide structural and kinetic insights into the signaling mechanism of aureochromes. The slow response points to an association of LOV to bZIP in the dark state prior to activation.

Identification of promoter targets by Aureochrome 1a in the diatom Phaeodactylum tricornutum

“…7 ). Specifically, it has been reported that under BL illumination, the Jα-helix influences the dimerizations of the bZIP domains, confirming that the bZIP dimer cannot be formed without this unfolding of the Jα-helix in BL conditions ( Herman et al , 2013 ; Goett-Zink et al , 2023 ) and, further, the A'α-helix influences the LOV–LOV domain dimerization ( Herman and Kottke, 2015 ; Goett-Zink et al , 2023 ). Consequently, these structural changes in the LOV domain affect the bZIP domain’s flexibility and bZIP dimerization capacity, influencing PtAUREO1a’s gene regulation property.…”

“…As it is postulated that AUREOs evolved from a non-light-sensitive form to a light-sensitive form ( Khamaru et al , 2022a , Preprint), gene regulation of PtAUREO1a in the absence of BL illumination (RL or dark) might be indicative of its ancestry, and PtAUREO1a may have further evolved to serve comprehensive roles utilizing the sensory domain. Indeed, several studies have confirmed that the conformational changes of PtAUREO1a are influenced by BL illumination, specifically due to the changes in structure of the LOV domain ( Herman et al , 2013 ; Banerjee et al , 2016b ; Heintz and Schlichting, 2016 ; Goett-Zink et al , 2023 ). Although it is confirmed that PtAUREO1a can dimerize in the dark ( Banerjee et al , 2016b ; Heintz and Schlichting, 2016 ), BL induces the LOV domain to disassociate from the bZIP domain and induces a structural change of flanking helices of the LOV domain ( Goett-Zink et al , 2023 ) ( Fig.…”

“…Indeed, several studies have confirmed that the conformational changes of PtAUREO1a are influenced by BL illumination, specifically due to the changes in structure of the LOV domain ( Herman et al , 2013 ; Banerjee et al , 2016b ; Heintz and Schlichting, 2016 ; Goett-Zink et al , 2023 ). Although it is confirmed that PtAUREO1a can dimerize in the dark ( Banerjee et al , 2016b ; Heintz and Schlichting, 2016 ), BL induces the LOV domain to disassociate from the bZIP domain and induces a structural change of flanking helices of the LOV domain ( Goett-Zink et al , 2023 ) ( Fig. 7 ).…”

“… Simplified working model of PtAUREO1a’s functional mechanism under dark and blue light conditions. This working model is adapted from Goett-Zink et al (2023) with modifications. (A) A PtAUREO1a dimer bound to the DNA in the absence of blue light illumination, most likely under dark or red light conditions.…”