Protein–chromophore interactions controlling photoisomerization in red/green cyanobacteriochromes

Abstract

Photoreceptors in the phytochrome superfamily use 15,16-photoisomerization of a linear tetrapyrrole (bilin) chromophore to photoconvert between two states with distinct spectral and biochemical properties. Canonical phytochromes include master regulators of plant growth and development in which light signals trigger interconversion between a red-absorbing 15Z dark-adapted state and a metastable, far-red-absorbing 15E photoproduct state. Distantly related cyanobacteriochromes (CBCRs) carry out a diverse range of photoregulatory functions in cyanobacteria and exhibit considerable spectral diversity. One widespread CBCR subfamily typically exhibits a red-absorbing 15Z dark-adapted state similar to that of phytochrome that gives rise to a distinct green-absorbing 15E photoproduct. This red/green CBCR subfamily also includes red-inactive examples that fail to undergo photoconversion, providing an opportunity to study protein–chromophore interactions that either promote photoisomerization or block it. In this work, we identified a conserved lineage of red-inactive CBCRs. This enabled us to identify three substitutions sufficient to block photoisomerization in photoactive red/green CBCRs. The resulting red-inactive variants faithfully replicated the fluorescence and circular dichroism properties of naturally occurring examples. Converse substitutions restored photoconversion in naturally red-inactive CBCRs. This work thus identifies protein–chromophore interactions that control the fate of the excited-state population in red/green cyanobacteriochromes.

Elucidating the origins of phycocyanobilin biosynthesis and phycobiliproteins

“…This construct was combined with Synechocystis ho1 ( sll1184 ) or with the POZ53545 HO from Methylovulum psychrotolerans Sph1, recently reported to improve coproduction of BV in E. coli [ SI Appendix , Fig. S6 A ; ( 39 )]. Each plasmid was tested by coexpression with the CBCR Ava_3771.…”

“…S13 C ), with comparison of the absorption and excitation spectra indicating possible heterogeneity. We used the ratio method ( 37 , 39 ) with the commercially available dye Alexa647 to estimate the quantum yield of MBL9008307 at 3% with PCB chromophore ( SI Appendix , Fig. S13 D ).…”

“…For reconstitution of PCB biosynthesis, we used plasmid Spam-545/PcyA as a starting point. Spam-545/PcyA was constructed from pKT271 ( 35 , 39 ) by introducing additional BamHI and SpeI restriction sites to generate Spam-pKT ( SI Appendix , Fig. S6 A ).…”

Biliverdin incorporation into the cyanobacteriochrome SPI1085g3 from Spirulina

“…Furthermore, photoconversion of miRFP670nano and NpR2164g5 ( Rockwell et al, 2012 ) could not be detected, implying a solid dark-adapted state and an extremely low quantum yield for photoconversion. They could be classified into a group of red-inactive CBCRs ( Rockwell et al, 2022 ). Due to the CY motif, C448S_CY exhibited an increased BV-binding efficiency, a relatively large Stokes shift, and significantly enhanced fluorescence.…”

“…They require only a GAF (cGMP-specific phosphodiesterase/adenylate cyclase/FhlA) domain for the incorporation of a linear tetrapyrrole serving as a sensory module, among which red/green CBCRs that utilize phycocyanobilin (PCB) to detect red light in most cases have been deeply studied and characterized ( Fushimi and Narikawa, 2019 ). The typical red/green CBCRs bind to a PCB chromophore via a conserved canonical Cys residue within their GAF domains and show reversible photoconversion between the red-absorbing dark-adapted state and the green-absorbing photoproduct state, which is triggered by Z/E isomerization of a double bond between the C15 and C16 positions of the chromophore ( Narikawa et al, 2008 ; Zhang et al, 2010 ; Rockwell et al, 2012 , 2015 , 2022 ; Pennacchietti et al, 2015 ; Wiebeler et al, 2019 ; Xu et al, 2020 ; Kirpich et al, 2021 ; Rao et al, 2021 ; Song et al, 2021 ). These domains are considered excellent candidates for bioimaging applications due to their small molecular size, natural monomeric state, remarkable spectral diversity, and especially interesting photoswitching behavior.…”