The Red‐/Green‐Switching GAF3 of Cyanobacteriochrome Slr1393 from Synechocystis sp. PCC6803 Regulates the Activity of an Adenylyl Cyclase

Abstract

Cyanobacteriochromes (CBCRs) are photoreceptors in cyanobacteria that present a bilin chromophore-binding GAF domain as a photochromic element to control the activity of a downstream enzyme or regulator. CBCR Slr1393 from Synechocystis PCC 6803 carries three GAF domains, but only the third one binds phycocyanobilin covalently. Slr1393 shows photochromicity between red and green absorbing states and regulates a C-terminally located histidine kinase. In this work, we fused this third GAF domain to an adenylyl cyclase (AC) from Microcoleus chthonoplastes PCC7420 that in its genuine form is under blue-light control from a LOV domain. A series of RGS-AC variants were constructed with various lengths of the linkers between RGS and AC. Assays in vitro and in living Escherichia coli cells (AC-deletion mutant) demonstrated that the activity of AC was light regulated, namely, the red-light-converted form of RGSΔ14-Δ4AC (in vitro) was about three times more active than the green-light-converted form. Expression of the fusion protein RGSΔ14-Δ4AC in vivo again showed highest light regulation with at least threefold amplification of the AC function. In some experiments, even tenfold higher activity was observed, which indicated that the protein, if expressed under in vivo conditions, was part of the E. coli physiological conditions and thereby subjected to more complex and variable regulation through other E. coli inherent factors.

Nanosecond protein dynamics in a red/green cyanobacteriochrome revealed by transient IR spectroscopy

“…CBCR PSMs are becoming increasingly popular as building blocks for optogenetic tools, because they can be fused to a variety of catalytic domains to allow for allosteric photocontrol of their respective reactions. To name a few examples, CBCRs recently have been engineered to function as adenylyl cyclases [4,5], to control protein-protein interactions [6], and gene expression in bacteria [7,8]. Furthermore, the small size, spectral diversity and fluorescence properties of CBCR PSMs are promising for applications as markers in fluorescence multiplexing and super-resolution microscopy [9].…”

“…This protein domain binds PCB and is located in a sequence after two non-photoactive GAF domains (counted from the N-terminus), therefore denoted Slr-g3 in the following. Slr1393 naturally acts as a light-regulated histidinee kinase, but by artificially fusing Slr-g3 to an adenylyl cyclase, photocontrol over this domain was also achieved [5]. Slr-g3 is a small protein domain (190 aa, 22 kDa) that converts reversibly between a red-light absorbing (Pr) and a green-light absorbing (Pg) parent state [20].…”

MAS NMR on a Red/Far-Red Photochromic Cyanobacteriochrome All2699 from Nostoc

“…Several outstanding features make CBCR-GAF domains interesting research objects: Their small size, high photochemical stability, large extinction coefficient and fluorescence quantum yields up to 15%. Moreover, they are able to modulate the activities of various enzyme modules to which they have been fused translationally, rendering them promising tools for optogenetic applications [21,22]. The second significant feature of CBCRs compared to canonical phytochromes is their wide range of absorption maxima of dark and photoproduct states.…”

Red, Orange, Green: Light- and Temperature-Dependent Color Tuning in a Cyanobacteriochrome

“…9 This output module can be exchanged by an adenylyl cyclase domain, producing a valuable optogenetic tool. 10 The third GAF domain of Slr1393 (denoted Slr1393-g3) binds a phycocyanobilin (PCB) chromophore 11 via a thioether bridge to a cysteine residue (C528) and interconverts between red-and green-absorbing parent states (Pr and Pg, respectively). 9 Slr1393-g3 has recently been crystallized in the Pr and Pg forms and in an intermediate state.…”

“…PCC6803, is a photosensory protein that consists of three GAF domains followed by a PAS domain and a signaling C-terminal histidine kinase (HK) domain . This output module can be exchanged by an adenylyl cyclase domain, producing a valuable optogenetic tool . The third GAF domain of Slr1393 (denoted Slr1393-g3) binds a phycocyanobilin (PCB) chromophore via a thioether bridge to a cysteine residue (C528) and interconverts between red- and green-absorbing parent states (Pr and Pg, respectively) .…”