Fused‐Gene Approach to Photoswitchable and Fluorescent Biliproteins

Abstract

Fluorescent and photoswitchable proteins are invaluable in life sciences and considered for applications in data storage. Of particular interest for in vivo studies are fluorescent proteins whose chromophores are generated autocatalytically from the amino acid chain; [1] some of them can also be switched between two states. [2,3] Alternatively, apoproteins can be used that spontaneously incorporate endogenous chromophores like retinal. [4,5] The open-chain tetrapyrrole chromophore of biliproteins is subject to remarkable excited-state control of the chromophore by the apoprotein. [6][7][8] Absorption and fluorescence of free bilins like the phycocyanobilin (PCB) is strongly increased in native biliproteins: the maximum can be shifted by over 100 nm, and a photochemical reaction path is opened in photochromic biliproteins like phytochromes [9] and cyano-(bacterio)chromes. [7] These natural variations and the possibility to modulate the photophysical properties render biliproteins, in principle, excellent biomarkers and photonic materials.Applications have been limited, however, because the bilin chromophores must be provided separately and then attached covalently to the apoproteins. Previously, genes of the apoprotein were co-expressed with genes whose products generate the bilin chromophore from endogenous heme and then attach it covalently to the apoprotein. [10][11][12] We now report an alternative approach that generates various biliproteins in situ from a single, multifunctional gene and endogenous heme. This approach is demonstrated by the synthesis of two persistently red-fluorescent biliproteins based on allophycocyanins, and by photochromic biliproteins derived from a novel cyanobacteriochrome that can be reversibly switched from a state absorbing and strongly fluorescing in the red, to a spectroscopically well-separated, less fluorescent state absorbing in the green spectral region.Gene slr1393 of the cyanobacterium Synechocystis sp. PCC6803 encodes a red-green photoreversible cyanobacteriochrome. The full-length protein contains three GAF domains, but GAF3 (aa 441-596) alone is capable of autocatalytically binding PCB to cysteine-528.[21] Addition of PCB to GA results in a reversibly photochromic chromoprotein, termed RGS (red-green switchable protein): state P r (l max = 650 nm) is strongly fluorescent (F F = 0.06); it is reversibly converted by irradiation with red light into state P g (l max = 539 nm), which has reduced and strongly blueshifted fluorescence (Table 1, Figure 1 a). Photoswitching can be repeated many times; it is stable over a wide pH range, and is retained after RGS is embedded into polyvinyl alcohol (PVA) film (see Figures S1 and S2 in the Supporting Information).Chromophorylated RGS can be produced in E. coli [11,13] that has been multiply transformed to produce the GAF3 apoprotein and two biosynthetic enzymes generating PCB from heme, that is, heme oxygenase (HO1) and the biliverdin reductase (PcyA). The cells show an intense red fluorescence that can be abolished by irradiation w...

Near-UV cyanobacteriochrome signaling system elicits negative phototaxis in the cyanobacterium Synechocystis sp. PCC 6803

“…Like those present in other cyanobacteria, multiple photosensors that could serve this function are representatives of the cyanobacterial phytochrome, CBCR, BLUF, and CRY families. Of particular note, the blue-absorbing CRY-related cyanopterin Sll1629 (9), the BLUF protein SyPixD/ Slr1694 (8), the red/green CBCR Slr1393 (38), and the red/far-red light sensor Cph1 (39) all could contribute to sustaining the signaling gradient necessary for LsiR-dependent (and potentially LsiR-independent) negative phototaxis at elevated fluence rates of visible light. This work also illuminates the exquisite complexity with which cyanobacteria tune not only their metabolism but their behavior in response to light and other environmental cues.…”

Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin

“…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.…”

“…9 PEB-All2699 GAF1, PEB(PUB)-All1280 GAF2 and PEB (PUB)-Slr1393 GAF3 are strongly fluorescent ( Table 1). The fusion with ho1 and pebS was successful in all2699 gaf1::ho1:: pebS, all1280 gaf2::ho1::pebS and slr1393 gaf3::ho1::pebS (Fig.…”

“…[5][6][7][8][9] There are several advantages of Phys 10 and CBCRs, 1 or domains thereof, that make them potentially interesting tools in biological analytics: they can be obtained by heterologous co-expression of protein-and chromophorecoding genes; and chromophore attachment is autocatalytic and does not require co-expression of lyases. 11 Their use as fluorescent biomarkers is impaired, however, by the relatively low fluorescence quantum yield.…”

Modular generation of fluorescent phycobiliproteins

“…[19][20][21] A somewhat simplified approach reducing the number of plasmids required for transformants capable of producing biliproteins has recently been demonstrated. 22 It is based on expressing the structural gene fused to genes coding for chromophore biosynthesis from endogenous heme. It requires the introduction of only a single gene for labeling and yields fluorescent proteins, which, however, as a disadvantage, exhibit a rather large size due to the fusion approach.…”