The Cruciality of Single Amino Acid Replacement for the Spectral Tuning of Biliverdin-Binding Cyanobacteriochromes

Abstract

Cyanobacteriochromes (CBCRs), which are known as linear tetrapyrrole-binding photoreceptors, to date can only be detected from cyanobacteria. They can perceive light only in a small unit, which is categorized into various lineages in correlation with their spectral and structural characteristics. Recently, we have succeeded in identifying specific molecules, which can incorporate mammalian intrinsic biliverdin (BV), from the expanded red/green (XRG) CBCR lineage and in converting BV-rejective molecules into BV-acceptable ones with the elucidation of the structural basis. Among the BV-acceptable molecules, AM1_1870g3_BV4 shows a spectral red-shift in comparison with other molecules, while NpF2164g5_BV4 does not show photoconversion but stably shows a near-infrared (NIR) fluorescence. In this study, we found that AM1_1870g3_BV4 had a specific Tyr residue near the d-ring of the chromophore, while others had a highly conserved Leu residue. The replacement of this Tyr residue with Leu in AM1_1870g3_BV4 resulted in a blue-shift of absorption peak. In contrast, reverse replacement in NpF2164g5_BV4 resulted in a red-shift of absorption and fluorescence peaks, which applies to fluorescence bio-imaging in mammalian cells. Notably, the same Tyr/Leu-dependent color-tuning is also observed for the CBCRs belonging to the other lineage, which indicates common molecular mechanisms.

Computational identification of key residues regulating fluorescence emission in a red/green cyanobacteriochrome

“…Moreover, BV is naturally synthesized in the process of heme degradation and is therefore available in sufficient quantities in many organisms, including mammals 5,20,21 . Despite several bacterial PHYs with BV chromophores have been engineered as near‐infrared (NIR) fluorescent proteins, CBCRs have the added advantage of exhibiting diverse spectral properties with single domain requirement which have attracted attention as a new imaging tool 11,19,20,22–25 . The recombinant AM1_1557g2 of red/green type CBCR from the chlorophyll d–bearing cyanobacterium Acaryochloris marina that photoconverts between far‐red (Pfr) and orange (Po) was the first reported CBCR to bind both PCB and BV but showed very low fluorescence 15 .…”

“…5,20,21 Despite several bacterial PHYs with BV chromophores have been engineered as near-infrared (NIR) fluorescent proteins, CBCRs have the added advantage of exhibiting diverse spectral properties with single domain requirement which have attracted attention as a new imaging tool. 11,19,20,[22][23][24][25] The recombinant AM1_1557g2 of red/green type CBCR from the chlorophyll d-bearing cyanobacterium Acaryochloris marina that photoconverts between far-red (Pfr) and orange (Po) was the first reported CBCR to bind both PCB and BV but showed very low fluorescence. 15 Considering this, engineered BVbinding CBCRs with red-shifted absorbance and fluorescence will be a powerful tool as an efficient NIR fluorescent protein.…”

Protein–chromophore interactions controlling photoisomerization in red/green cyanobacteriochromes

“…CBCRs have diversified into a range of subfamilies that can detect light from 378 -741 nm via a range of tuning mechanisms [49,54,55,65,66,81,[87][88][89][90][91][92][93]. Prototypical members of one abundant CBCR subfamily, the red/green CBCRs, exhibit a red-absorbing 15Z PCB chromophore in the dark-adapted state (Fig.…”

Biliverdin incorporation into the cyanobacteriochrome SPI1085g3 from Spirulina

“…bind not only PCB but also biliverdin (BV, Figure 6A ; Narikawa et al, 2015a , b ; Fushimi et al, 2016 ; Blain-Hartung et al, 2018 ; Moreno et al, 2020 ), which is abundant in many potential host cells, including mammalian tissues, and represents a longer wavelength-absorbing chromophore to facilitate deeper penetration into the tissues ( Chen et al, 2021 ; Manoilov et al, 2022 ). Therefore, more attention has been given to PCB replacement with BV to incorporate CBCRs for advantages in applications ( Fushimi et al, 2019 , 2020 ; Oliinyk et al, 2019 ; Buhrke, 2021 ; Tachibana et al, 2021 ; Tang et al, 2021 ; Ma et al, 2022 ).…”