Green, orange, red, and far-red optogenetic tools derived from cyanobacteriochromes

Abstract

Existing optogenetic tools for controlling protein-protein interactions are available in a limited number of wavelengths thereby limiting opportunities for multiplexing. The cyanobacteriochrome (CBCR) family of photoreceptors responds to an extraordinary range of colors, but light-dependent binding partners for CBCR domains are not currently known. We used a phage-display based approach to develop small (~50-residue) monomeric binders selective for the green absorbing state (Pg), or for the red absorbing state (Pr) of the CBCR Am1_c0023g2 with a phycocyanobilin chromophore and also for the far-red absorbing state (Pfr) of Am1_c0023g2 with a biliverdin chromophore. These bind in a 1:1 mole ratio with KDs for the target state from 0.2 to 2 µM and selectivities from 10 to 500-fold. We demonstrate green, orange, red, and far-red light-dependent control of protein-protein interactions in vitro and also in vivo where these multicolor optogenetic tools are used to control transcription in yeast.

Needles in a haystack: H-bonding in an optogenetic protein observed with isotope labeling and 2D-IR spectroscopy

“… 1–3 This class of proteins recently recieved increasing attention as building blocks for optogenetic tools because their modular domain structure allows to light-trigger cellular responses in different target organisms. 4–7 One example is the light-regulated expression of target genes, which was recently achieved in yeast with a two-component optogenetic system 8 based on the red/green CBCR Am1-c0023g2 (from here abbreviated AmI-g2) from the cyanobacterium Acarychloris marina . 9 AmI-g2 is a member of the red/green lineage of CBCRs and can incorporate two different light-sensitive chromophores: phycocyanobilin (PCB) or biliverdin.…”

The rise and shine of yeast optogenetics

“…Furthermore, the Bam-green1.3 protein was identified as the interacting partner of Am1_c0023g2, when this latter protein is bound to PCB and upon green-light stimulation (Jang et al, 2019). Thus, the Am1_c0023g2 and Bam-green1.3 interaction was used for light-controlled β-galactosidase expression in yeast (Jang et al, 2019). This example shows the potential of cyanobacteria photoreceptors in the future expansion of the current set of optogenetic switches available in yeast.…”

“…The CBCRs contain GAF domains (cGMP-phosphodiesterase/adenylate cyclase/FhlA), which respond to light and generally bind PCB (Anders & Essen, 2015). Recently, the GAF domain of a CBCR (Am1_c0023g2) from Acaryochloris marina was used in yeast optogenetics (Jang, McDonald, Uppalapati, & Woolley, 2019). This CBCR has the capacity of red/green reversible photoconversion and the possibility to bind PCB or biliverdin chromophores (Fushimi et al, 2016).…”

“…This CBCR has the capacity of red/green reversible photoconversion and the possibility to bind PCB or biliverdin chromophores (Fushimi et al, 2016). Importantly, when Am1_c0023g2 binds PCB, it is capable of red/green photoconversion, and when it binds biliverdin, far-red/orange photoconversion was observed (Fushimi et al, 2016;Jang et al, 2019). Furthermore, the Bam-green1.3 protein was identified as the interacting partner of Am1_c0023g2, when this latter protein is bound to PCB and upon green-light stimulation (Jang et al, 2019).…”

“…Importantly, when Am1_c0023g2 binds PCB, it is capable of red/green photoconversion, and when it binds biliverdin, far-red/orange photoconversion was observed (Fushimi et al, 2016;Jang et al, 2019). Furthermore, the Bam-green1.3 protein was identified as the interacting partner of Am1_c0023g2, when this latter protein is bound to PCB and upon green-light stimulation (Jang et al, 2019). Thus, the Am1_c0023g2 and Bam-green1.3 interaction was used for light-controlled β-galactosidase expression in yeast (Jang et al, 2019).…”