Thiol-Based Photocycle of the Blue and Teal Light-Sensing Cyanobacteriochrome Tlr1999

Abstract

Cyanobacteriochromes are a spectrally diverse photoreceptor family that binds a bilin chromophore. For some cyanobacteriochromes, in addition to the widely conserved cysteine to anchor the chromophore, its ligation with a second cysteine is responsible for a remarkable blue shift. Herein, we report a newly discovered cyanobacteriochrome Tlr1999 exhibiting reversible photoconversion between a blue-absorbing form at 418 nm (P418) and a teal-absorbing form at 498 nm (P498). Acidic denaturation suggests that P418 harbors C15-Z phycoviolobilin, whereas P498 harbors C15-E phycoviolobilin. When treated with iodoacetamide, which irreversibly modifies thiol groups, P418 is slowly converted to a green-absorbing photoinactive form denoted P552. The absorption spectrum of P498 appears to be unaffected by iodoacetamide, but when iodoacetamide modified, it is photoconverted to P552. These results suggest that a covalent bond exists between the second Cys and the phycoviolobilin in P418 but not in P498. Subsequent treatment with dithiothreitol converts P552 into P418, whereas dithiothreitol reduces P498 to yield P420, a photoinactive form. Site-directed mutagenesis shows that the second Cys is essential for assembly of the photoactive holoprotein and that the photoactivity of this inert mutant is partially rescued by β-mercaptoethanol. These results suggest that the covalent attachment and detachment of a thiol, although not necessarily that of the second Cys, is critical for the reversible spectral blue shift and the complete photocycle. We propose a thiol-based photocycle, in which the thiol-modified P552 and P420 are intermediate-like forms.

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

“…Our study of the protochromic photocycle and previous analyses of two-Cys photocycles (21,23,25,26,29) illustrate a common molecular theme for CBCRs: bilin photoisomerization does not itself induce a spectral change, but rather triggers a subsequent light-independent chemical reaction that induces a dramatic spectral shift. Interestingly, the secondary chemical reaction requires only a few residues within the GAF domain (e.g., the protochromic triad or the second Cys), providing a powerful mechanism for evolution of CBCRs with distinct spectral properties.…”

“…* 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.…”

“…CBCR subfamilies that sense light in the near-UV to blue region (330-470 nm) have been studied extensively. Such CBCRs combine bilin photoisomerization with subsequent formation or elimination of a second thioether linkage to the bilin C10 atom, shortening the conjugated π system to induce a remarkable spectral shift (19,21,23,(25)(26)(27)(28)(29). In such "two-Cys photocycles," the reactive thiol group is supplied by a second conserved Cys residue within the GAF domain.…”

Diatom Phytochromes Reveal the Existence of Far-Red-Light-Based Sensing in the Ocean

“…Sequences were cloned into pET28a (Novagen) in NcoI/XhoI sites for the full-length Pt-DPH gene and NcoI/SalI for the PSM of Pt-DPH and Tp-DPH, and expressed as C-terminal His6 tagged proteins in E. coli BL21 strain containing either the pKT270 plasmid for BV IXa, pKT272 for PFB, or pKT278 for PEB production (Mukougawa et al, 2006). Recombinant proteins were expressed with 0.1 mM (Pt-DPH) or 0.5 mM (Tp-DPH) isopropyl b-D-1-thiogalactopyranoside at 18°C overnight and purified and analyzed as described (Enomoto et al, 2012). Light-emitting diodes were used for R (639 nm for Pt-DPH and 690 nm for Tp-DPH) and FR (757 nm for Pt-DPH and 765 nm for Tp-DPH) light irradiations (halfbandwidths below 24 nm for the different wavelengths and fluences of ;20 and ;3 µmol photons$m 22 $s 21 for R and FR irradiation, respectively).…”

“…Difference spectra were calculated as "FR-irradiated" minus "R-irradiated" spectra. Proteins denaturation was done in acidic urea (pH 2) (Enomoto et al, 2012). Pt-DPH-FL and Tp-DPH-PSM dark relaxation was followed during 24 h at room temperature starting from DPH purified under room lighting.…”