Photophysical diversity of two novel cyanobacteriochromes with phycocyanobilin chromophores: photochemistry and dark reversion kinetics

Abstract

Cyanobacteriochromes are phytochrome homologues in cyanobacteria that act as sensory photoreceptors. We compare two cyanobacteriochromes, RGS (coded by slr1393) from Synechocystis sp. PCC 6803 and AphC (coded by all2699) from Nostoc sp. PCC 7120. Both contain three GAF (cGMP phosphodiesterase, adenylyl cyclase and FhlA protein) domains (GAF1, GAF2 and GAF3). The respective full-length, truncated and cysteine point-mutated genes were expressed in Escherichia coli together with genes for chromophore biosynthesis. The resulting chromoproteins were analyzed by UV-visible absorption, fluorescence and circular dichroism spectroscopy as well as by mass spectrometry. RGS shows a red-green photochromism (k max = 650 and 535 nm) that is assigned to the reversible 15Z ⁄ E isomerization of a single phycocyanobilin-chromophore (PCB) binding to Cys528 of GAF3. Of the three GAF domains, only GAF3 binds a chromophore and the binding is autocatalytic. RGS autophosphorylates in vitro; this reaction is photoregulated: the 535 nm state containing E-PCB was more active than the 650 nm state containing Z-PCB. AphC from Nostoc could be chromophorylated at two GAF domains, namely GAF1 and GAF3. PCB-GAF1 is photochromic, with the proposed 15E state (k max = 685 nm) reverting slowly thermally to the thermostable 15Z state (k max = 635 nm). PCB-GAF3 showed a novel red-orange photochromism; the unstable state (putative 15E, k max = 595 nm) reverts very rapidly (s 20 s) back to the thermostable Z state (k max = 645 nm). The photochemistry of doubly chromophorylated AphC is accordingly complex, as is the autophosphorylation: E-GAF1 ⁄ E-GAF3 shows the highest rate of autophosphorylation activity, while E-GAF1 ⁄ Z-GAF3 has intermediate activity, and Z-GAF1 ⁄ Z-GAF3 is the least active state. Structured digital abstractl AphC phosphorylates AphC by protein kinase assay (View interaction) l RGS phosphorylates RGS by protein kinase assay (View interaction) Abbreviations AphC, protein encoded by aphC = all2699; CBR, cyanobacteriochrome; GAF, cGMP phosphodiesterase, adenylyl cyclase and FhlA protein domain (SMART acc. no. SM00065); KPB, potassium phosphate buffer; Nostoc, Anabaena (Nostoc) sp. PCC 7120; P XXX ⁄ P YYY , the two photoconvertible states of CBR or Phy designated by the absorption maxima, with the stable generally 15Z state (k max = XXX nm) preceding the light-activated generally 15E-configured state (k max = YYY nm); PAS, period circadian protein, Ah receptor nuclear translocator protein and single-minded protein domain (SMART acc. no. SM00091); PCB, phycocyanobilin; Phy, phytochrome; PVB, phycoviolobilin; PFB, phytochromobilin; RGS, red-green switchable protein encoded by rgs = slr1393; Synechocystis, Synechocystis sp. PCC 6803.

Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin

“…1, S2 †). There are, however, exceptions: during the chromophorylation of Slr1393 GAF3 21 and Alr3356, 15 PCB does not isomerize, while part of PEB can isomerize to PUB (Fig. 1, S2 †).…”

“…31 However, PEBchromophorylated GAFs did not form well in E. coli cells following this protocol. 9,21 Changing to PebS was more effective, 22 as it generates PEB from biliverdin as a single enzyme. 32 The PebS-based approach allowed many GAFs of CBCRs to be chromophorylated with PEB ( Fig.…”

Green/red cyanobacteriochromes regulate complementary chromatic acclimation via a protochromic photocycle

“…* Cyanobacteriochromes (CBCRs) are widespread cyanobacterial photosensors with phytochrome-related GAF domains (1,2,13,14). Although CBCRs also convert between two photostates via bilin photoisomerization at C15, they exhibit much more spectral diversity, with peak absorptions ranging from 330 to 680 nm and hence spanning the entire visible spectrum and near UV (13,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). CBCR subfamilies that sense light in the near-UV to blue region (330-470 nm) have been studied extensively.…”

Cyanobacteriochromes in full color and three dimensions

“…However, the conserved aspartate plays different roles: in Cph1, it interacts with a conserved residue in the PHY domain, but in AnPixJ it directly interacts with the bilin rings A, B, and C (4). The structural basis for formation of the green-absorbing photoproduct of AnPixJ and related proteins remains to be elucidated (10,13,14). The case is reversed for TePixJ, in which the green-absorbing photoproduct was crystallized and the blue-absorbing dark state remains to be characterized.…”

“…CBCRs instead achieve fully reversible photochemistry with a lone chromophore-binding GAF domain. Multiple CBCRs often occur in tandem within a single protein, allowing integration of multiple light signals at a single C-terminal output domain (10). Whereas Phys predominantly respond to the red/far-red spectral region, CBCRs display a rich variety of photocycles spanning the entire visible and near-UV spectrum (2,(11)(12)(13).…”