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Identification of cyanobacteriochromes detecting far-red light Nathan Clarke Rockwell, Shelley S. Martin, and J. Clark Lagarias Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00299 • Publication Date (Web): 13 Jun 2016 Downloaded from http://pubs.acs.org on June 14, 2016

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Biochemistry

Identification of cyanobacteriochromes detecting far-red light Nathan C. Rockwell, Shelley S. Martin, and J. Clark Lagarias* Department of Molecular and Cellular Biology, University of California, Davis, CA 95616

Corresponding author: J. C. Lagarias, Department of Molecular and Cell Biology, 31 Briggs Hall, University of California at Davis, Davis CA 95616. Telephone: 530-7521865; FAX: 530-752-3085; E-mail: [email protected] † This work was supported by a grant from the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, United States Department of Energy (DOE DE-SC0002395 to J.C.L.).

Conflict of Interest Disclosure. The authors declare no competing financial interest.

Running head: Far-red cyanobacteriochromes

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Biochemistry

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Abstract The opacity of mammalian tissue to visible light and the strong attenuation of infrared light by water at ≥900 nm have contributed to growing interest in the development of farred and near-infrared absorbing tools for visualizing and actuating responses within live cells. Here we report the discovery of cyanobacteriochromes (CBCRs) responsive to light in this far-red window. CBCRs are linear tetrapyrrole (bilin)-based light sensors distantly related to plant phytochrome sensors. Our studies reveal far-red (max 725-755 nm)/orange (max 590-600 nm) and far-red/red (max 615-685 nm) photoswitches that are small (< 200 amino acids) and can be genetically reconstituted in living cells. Phylogenetic analysis and characterization of additional CBCRs demonstrated that farred/orange CBCRs evolved after a complex transition from green/red CBCRs known for regulating complementary chromatic acclimation (CCA). Incorporation of different bilin chromophores demonstrates that tuning mechanisms responsible for red-shifted chromophore absorption act at the A-, B-, and/or C-rings, whereas photoisomerization occurs at the D-ring. Two such proteins exhibited detectable fluorescence extending well into the near infrared. This work extends the spectral window of CBCRs to the edge of the infrared, raising the possibility of using CBCRs in synthetic biology applications in the far-red region of the spectrum.


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Footnotes [note a]. In describing photocycles, we use a convention whereby the photostate with the 15Z bilin configuration is listed first, followed by the photostate with the 15E configuration. Color definitions in this study are near-UV, 300-394 nm; violet, 395-410 nm; blue, 411-485 nm; teal, 486-514 nm; green, 515-569 nm; yellow, 570-585 nm; orange, 586-614 nm; red, 615-685 nm; far-red, 686-760 nm; near-infrared, 761-1000 nm. [note b]. Abbreviations: BV, biliverdin IX; CBCR, cyanobacteriochrome; CCA, complementary chromatic acclimation; DXCF, Asp-Xaa-Cys-Phe; FaRLiP, far-red lightinduced photoacclimation; GAF: cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and formate hydrogen lyase transcription activator FhlA; PCB, phycocyanobilin; PEB, phycoerythrobilin; PB, phytochromobilin; PHY, phytochromespecific domain.


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Almost all organisms use photosensory proteins to sense the ambient light environment and to tune their metabolism and behavior. Animal photoreceptors provide a basis for diverse biological responses including the entrainment of circadian rhythms and visual navigation.[1, 2] Photosynthetic organisms also utilize diverse photosensors.[3] For example, flavin-based phototropins control plant phototropism, a photobiological response first noted in antiquity and studied by Charles Darwin.[4-6] Plants also contain phytochromes, linear tetrapyrrole (bilin)-containing sensors that measure red and far-red light[note a] to control many aspects of plant biology, from seed germination and lightdependent growth and development (photomorphogenesis) to shade avoidance and flowering.[7-10]

Photosynthetic

and

nonphotosynthetic

bacteria

also

contain

photoreceptors.[11-13] Indeed, the first photobiological response discovered in cyanobacteria, complementary chromatic acclimation (CCA),[note b] was reported within 25 years of Darwin‟s studies on phototropism[14] and is now known to leverage bilinbased photoreceptors to optimize light harvesting under green or red light.[15-17] More recently, photoproteins have become critical research tools. Cell biology has been profoundly altered by the discovery and development of green fluorescent protein,[18] and light-dependent channelrhodopsins have proven equally transformational in the development of optogenetic approaches to neurobiology.[19] Phytochromes have also attracted attention as fluorescent and photoacoustic probes,[20-26] as reagents for controlling protein-protein interactions with light,[27] in systems for light-controlled gene expression,[28] and as tools for regulation of second messenger metabolism with light.[29, 30] Phytochromes are particularly appealing for application in multicellular animals due to their peak absorption in the red to far-red, partially overlapping the far-


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Biochemistry

red/near-infrared (near-IR) window of optimum transparency in animal tissues.[31] Metazoans also lack phytochromes, so there is no endogenous phytochrome photobiology in animals. However, counterbalancing points limit such applications of phytochromes. The minimal size for photochemically and biologically functional phytochromes is relatively large (300-500 amino acids), phytochromes are often dimeric, and many phytochromes utilize reduced linear tetrapyrrole (bilin) chromophores not present in animal cells.[7, 32, 33] Moreover, most phytochromes exhibit an unusual knotted architecture [34] that can constrain their application as C-terminal tags or reporters in fusion constructs. Cyanobacteriochromes (CBCRs) present a possible alternative. Like the distantly related phytochromes, CBCRs use 15,16-photoisomerization of bilin chromophores (Fig. 1) to reversibly photoconvert between two states with distinct spectral and biochemical properties.[33, 35] In both CBCRs and phytochromes, the bilin is covalently attached to a conserved Cys residue via a thioether linkage (Fig. 1). The minimal CBCR photosensory domain is much smaller than that of phytochromes (70 nm in the native protein. Moreover, some far-red/orange CBCRs exhibit detectable far-red and near-infrared fluorescence. Our studies establish far-red CBCRs as promising new lead compounds for diverse applications in live cells and provide new insight into detection of far-red and near-IR light by bilin chromophores. MATERIALS AND METHODS Bioinformatics. CBCR sequences were identified using BLAST searches[57] against the Genbank and DOE-IMG databases. Locus tags from DOE-IMG are reported in Tables S1-S2. All phylogenies were calculated using maximum-likelihood methods with structural information in PhyML-structure.[58] To generate the final alignment used for calculating the CBCR phylogeny presented in Fig. 2, new CBCR sequences were manually added to a pre-existing alignment.[53] The resulting alignment was pruned, and the region encompassing the Asp-motif was manually adjusted to optimize conservation of hydrophobic residues. Key regions are presented in Fig. S1. Structural information was projected onto the sequence alignment using an in-house script as described[53] with CBCR crystal structures for TePixJ and AnPixJ (PDB accession codes 3W2Z, 4FOF, and 4GLQ).[59, 60] TePixJ and AnPixJ themselves are not associated with histidine kinases and hence were removed for final phylogeny calculation, because the encoding of the structural information in PhyML-structure is not tied to the individual sequences.[58] Cterminal His kinase regions were initially aligned to sequences from histidine kinases for which crystal structures were available (PDB accession codes: 2C2A, 3DGE, 4U7N,


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4U7O, 3D36, 4R39) [61-64] using MUSCLE.[65] The resulting alignment was adjusted manually and structural information was added to the alignment using the in-house script as described above to yield the alignment used for calculating the maximum-likelihood phylogeny presented in Fig. 2. Key regions are presented in Fig. S2. Sequences for the crystal structures were again removed prior to calculation of the phylogeny, because those sequences were not associated with CBCR domains and hence would not be matched in the „tanglegram‟ representation of Fig. 2. Both phylogenies were calculated in PhyML-structure using the six-matrix EX_EHO model in partitioning mode, using the LG substitution matrix for positions with no structural information.[58] Maximum likelihood estimates were used for the proportion of invariable sites and for the distribution of the gamma shape parameter, with four substitution categories and optimization of tree topology, branch length, and rate parameters.

The

resulting

tree

was

processed

using

FigTree

(available

at

http://tree.bio.ed.ac.uk/software/figtree/) and graphics editing software. Cloning, expression, and purification of CBCRs. Anacy_4718g3 (amino acids 1274–1466 of the Anacy_4718 locus in Anabaena cylindrica PCC 7122), Anacy_2551g3 (amino acids 835–1026) of Anacy_2551), and Oscil6304_4080 (amino acids 341-515 of Oscil6304_4080 in Oscillatoria acuminata PCC 6304) were cloned from genomic DNA prepared from Anabaena sp. PCC 7938 and Oscillatoria acuminata PCC 6304 (generous gift of Elsie Campbell and Prof. Jack Meeks, UC Davis) using PCR with appropriate primers and with addition of one to two amino acids at the N terminus to create a start codon with an NcoI restriction site. For Anabaena sp. PCC 7938, amplified DNA


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Biochemistry

sequences were identical to those of A. cylindrica PCC 7122. Cyan7822_4053g2 (amino acids 903–1091 of Cyan7822_4053 in Cyanothece sp. PCC 7822), Nos7524_4790 (amino acids 932-1105 of Nos7524_4790 in Nostoc sp. PCC 7524), Sta7437_1656 (amino acids 696-871 of Sta7437_1656 from Staniera cyanosphaera PCC 7437), and WP_016871037 (amino acids 1246-1419 of UYKDRAFT_01008 from Fischerella thermalis PCC 7521) were obtained as synthetic genes (Genscript, Piscataway, NJ) codon-optimized for expression in E. coli. Anacy_4718g3, Cyan7822_4053g2, Nos7524_4790, and Oscil6304_4080 were cloned into pBAD-Cph1-CBD[66] using unique NcoI and SmaI sites, generating in-frame fusions to a C-terminal intein-CBD tag. Expression in E. coli strain LMG194 with co-production of PCB using pPL-PCB followed published procedures.[67] Co-production of PB and PEB was performed in the same way, but used pPL-PB and pAT-PebS, respectively.[22, 44] Proteins were purified on chitin resin (NEB) as previously described, with final dialysis into TKKG buffer (25 mM TES-KOH pH 7.8, 100 mM KCl, 10% (v/v) glycerol).[53, 66] Anacy_2551g3, Anacy_4718g3, Sta7437_1656, and WP_016871037 were cloned into pET28-RcaE[17] using unique NcoI and BamHI sites, thereby cloning each CBCR as an in-frame fusion to a C-terminal His tag. His-tagged proteins were expressed in E. coli strain C41[68] with co-production of PCB using pKT271[69] and were purified on purified on His-bind Ni2+-NTA resin (Novagen) using an imidazole gradient.[17, 41] Histagged proteins were dialyzed into 20 mM sodium phosphate (pH 7.5), 50 mM NaCl, 10% (v/v) glycerol and 1 mM EDTA. Purified proteins were analyzed by SDS-PAGE using standard procedures and apparatus (Bio-Rad) followed by semi-dry transfer to


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PVDF membranes, staining with amido black for visualizing total protein, and zinc blotting (Fig. S3).[44] Spectroscopic characterization of CBCRs. Absorption spectra were acquired on a Cary 50 spectrophotometer at 25°C. Photoconversion was triggered in the absorption cuvette using 728 nm LEDs (Sanyo) or using a xenon source equipped with band-pass interference filters (400±35 nm, 550±35 nm, 600±20 nm, 650±20 nm).[66] For WP_016871037, a red laser pointer (632.8 nm, 2 mW) was used. Fluorescence spectra were acquired on a

QM-6/2005SE fluorimeter equipped with

red-enhanced

photomultiplier tubes (Photon Technology International 814 Series). For denaturation assays, a 100 µl aliquot of protein was added to 1 ml of 7 M guanidinium chloride/1% HCl (v/v). Denatured samples were illuminated using the xenon lamp equipped with a 320 nm long-pass filter, and extinction coefficients were estimated from the denatured spectra as described previously[50] using the known extinction coefficients for PCB under

acid

denaturation

conditions.[70]

The fluorescence

quantum

yield

of

Anacy_2551g3 was estimated using the ratio method with Alexa 750 (Thermo Fisher) as the reference standard.[22] RESULTS Conserved far-red/orange and far-red/red CBCR lineages. Our previous phylogenetic analyses clustered CBCR domains Anacy_4718g3 and Anacy_2551g3 from the filamentous cyanobacterium Anabaena cylindrica PCC 7122 with green/red CBCRs.[53] These sequences diverge from those of canonical green/red CBCRs, particularly in the Asp-motif region associated with spectral tuning in many CBCR lineages.[17, 42, 53, 60,


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Biochemistry

71] We therefore assessed whether these sequences might be part of a previously uncharacterized CBCR lineage by using them as queries in BLAST [57] searches. This approach identified additional CBCR sequences containing similar variant Asp-motifs (Table S1). These CBCRs could readily be aligned with those of known green/red CBCRs (Fig. S1). Maximum-likelihood phylogenetic analysis demonstrated that these sequences, including Anacy_4718g3 and Anacy_2551g3, formed part of a distinct cluster (Fig. 2). CBCR domains in this cluster were part of larger signaling molecules with Cterminal histidine kinases (Table S1 & Fig. S2) and possessed diverse full-length domain architectures associated with multiple subfamilies of C-terminal histidine kinase “output” domains (Fig. 2). By contrast, coherent CBCR/kinase pairings were observed for the green/red CBCR RcaE and apparent orthologs associated with type III CCA (Fig. 2 and see Discussion). Taken together, these studies establish Anacy_4718g3 and Anacy_2551g3 as members of a new subfamily of CBCR photosensors associated with evolutionarily diverged signaling proteins. We next characterized Anacy_4718g3 in vitro after recombinant expression in E. coli engineered to produce PCB.[67] This protein exhibited reversible photoconversion between far-red-absorbing and orange-absorbing states exhibiting peak absorption at 740 nm and 590 nm, respectively (Fig. 3A & Table 1). Similar photocycles were observed for two constructs with different affinity tags (Figs. 3A & S4A-B), indicating that the different reagents employed in purifying His-tagged proteins or intein-CBD fusion proteins did not affect the far-red/orange photocycle. In both cases, the specific absorbance ratio (SAR, Table 1) was lower than that seen with red-absorbing CBCRs. [17, 45, 50] This may indicate the presence of contaminating apoprotein in current


Biochemistry

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preparations. Slow dark reversion from the orange-absorbing state to the far-redabsorbing state was observed, indicating that the far-red-absorbing state is dark-adapted and the orange-absorbing state is the photoproduct (Fig. S4C). Anacy_2551g3 exhibited a similar reversible photocycle (Fig. 3B), with the far-red-absorbing maximum at a slightly shorter wavelength (728 nm: Table 1). A third member of this cluster, Cyan7822_4053g2 from the unicellular cyanobacterium Cyanothece sp. PCC 7822, exhibited almost identical behavior to Anacy_2551g3 (Figs. 3C & S4D; Table 1). These results establish the existence of a cluster of CBCRs related to green/red CBCRs but exhibiting conserved, reversible far-red/orange photocycles. Phylogenetic analysis tentatively placed far-red/orange CBCRs as part of a larger lineage that is sister to the CCA photoreceptors CcaS and RcaE (Fig. 2). Within this lineage, one branch of CBCRs includes proteins with Asp-motifs very similar to those of CcaS and RcaE, such as PlpA from Synechocystis sp. PCC 6803[72] and Oscil6304_4080 from Oscillatoria acuminata PCC 6304 (Fig. S1). The other branch includes both the farred/orange CBCRs and other sequences. Some of these other sequences have Cys residues in or near the Asp-motif (Fig. S1), including Nos7524_4790 and Sta7437_1656. Such Cys residues can form a second covalent linkage to the chromophore, resulting in absorption of blue to ultraviolet light.[42, 44, 73] We therefore characterized additional CBCRs to explore the transition from green/red photocycles to far-red/orange photocycles. Oscil6304_4080 exhibited a dark-adapted state with peak absorption in the green region of the spectrum (Fig. S5A). Illumination with green light (550±35 nm) produced only


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Biochemistry

minimal photoconversion. The peak wavelength and lineshape of the green-absorbing state were similar to those of the green-absorbing states previously reported for the CCA regulator RcaE from Fremyella diplosiphon and for the green/blue CBCR Oscil6304_4336g2 from O. acuminata (Fig. S5B). Comparison of the normalized difference spectra for Oscil6304_4080 and RcaE shows much less depletion of the green state

for

Oscil6304_4080,

whose

red-absorbing

photoproduct

state

appeared

heterogeneous and bleached (Fig. S5C). By contrast, Nos7524_4790 exhibited reversible photoconversion between two photostates with peak absorption in the blue and red regions of the visible spectrum (Fig. S5D & Table 1). The red-absorbing state was similar to that of RcaE (Fig. S5E). Neither Oscil6304_4080 nor Nos7254_4790 exhibited detectable species with far-red absorption. Sta7437_1656 belongs to the second branch and exhibited reversible photoconversion between states with far-red and red peak absorption (Fig. 3D). The far-red state was very similar to that of Anacy_2551g3, whereas the red-absorbing state was blue-shifted relative to that of RcaE (Fig. S4E-F). As purified, WP_016871037 exhibited a mix of red- and far-red-absorbing species (Fig. 3E), with ready conversion of the far-red state to the red state but poor reversibility even after laser illumination (Fig. S4G). The farred/red photochemical difference spectrum for WP_016871037 was similar to that of Sta7437_1656 (Fig. S4H). Sta7437_1656 and WP_016871037 are part of a small cluster of CBCR domains associated with diverse domain architectures (Table S2). These results demonstrate the existence a second branch of far-red CBCRs with far-red/red photocycles (Fig. 2).


Biochemistry

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Spatial separation of photoconversion and spectral tuning in the far-red-absorbing chromophore. The far-red-absorbing 15Z states of these newly described CBCRs exhibited remarkable red shifts relative to previously known CBCRs (ca. 650-710 nm)[17, 41, 45, 47, 48, 50-52]. We therefore used an acid denaturation assay[73-77] to examine chromophore structure in far-red CBCRs. In this assay (Fig. S6A), samples in either photostate are denatured by dilution into concentrated guanidinium chloride. In the absence of native protein structure, 15E bilins are unidirectionally photoconverted to the 15Z configuration by white light, allowing assignment of the chemical configuration of the photostates. Different bilins have characteristic spectra under denaturing conditions, with different peak wavelengths and with different relative intensities for the long- and short-wavelength chromophore absorption bands in the ultraviolet to visible spectrum (Fig. S6B). Although it is possible for labile structural changes to be lost upon denaturation, this assay can provide tentative identification of bilin species.[44, 46, 73] Acid denaturation of Anacy_4718g3 resulted in loss of the native spectral features in both photostates and revealed the presence of a porphyrin side population that was also observed in fluorescence spectroscopy (see below). Contaminating porphyrin populations have also been observed in previous studies of heterologously expressed phytochromes, sometimes as covalent adducts.[22, 78] Photoconversion of denatured samples established the far-red-absorbing state as having the 15Z configuration and the orangeabsorbing state as having the 15E configuration (Fig. 4A-B). Denaturation analysis of Anacy_2551g3 and Cyan7822_4053g2 confirmed that the far-red-absorbing states of all three far-red/orange CBCRs adopted the 15Z configuration (Fig. S7A-B), albeit with varying amounts of contaminating porphyrin. The far-red/red CBCR Sta7437_1656


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Biochemistry

exhibited a 15Z far-red-absorbing photostate and 15E red-absorbing photoproduct, with notably less contaminating porphyrin (Fig. 4C-D). Nos7524_4790 exhibited a similar red-absorbing 15E photoproduct, in this case with a blue-absorbing 15Z dark state (Fig. S5E-F). As purified, WP_016871037 exhibited both far-red- and red-absorbing species, confirmed as a mix of 15Z and 15E bilin by acid denaturation (Fig. S7C). Photoconversion of this protein with far-red light resulted in incomplete formation of 15E bilin (Fig. S7D), assigning the red-absorbing state as the 15E photoproduct. The presence of 15E photoproduct in this preparation arose due to the combination of light exposure during purification and poor reverse photoconversion in this protein (see above). Estimation of the extent of photoconversion in both samples by comparison to reference spectra[17] allowed us to subtract a scaled photoproduct spectrum from the initial spectrum, resulting in a spectrum similar to that of Sta7437_1656 in the far-red-absorbing photostate (Fig. 3E). These results demonstrate that far-red CBCRs exhibit a conserved 15Z photostate with peak absorption in the far-red and with blue-shifted 15E photoproducts absorbing orange or red light. Far-red CBCRs thus are reversed relative to phytochromes, in which the red-absorbing 15Z Pr state is blue-shifted relative to the farred-absorbing 15E Pfr state.[33, 79, 80] The

photochemical

difference

spectrum

for

denatured

Anacy_4718g3

was

superimposable on that of the red/green CBCR NpR6012g4 (Fig. 5A). Recent characterization of NpR6012g4 using solution NMR spectroscopy confirmed the presence of a covalent PCB adduct in both photostates, with intramolecular nuclear Overhauser effect cross-peaks confirming photoisomerization at the 15,16-double bond.[38, 81] The peak wavelength and relative bilin band intensities of denatured 15Z


Biochemistry

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Anacy_4718g3 in the far-red state were also consistent with a 15Z covalent PCB adduct (Fig. 5B). This correlation plot also demonstrates a difference between covalent and noncovalent PCB species under denaturing conditions, and this assay has been used to demonstrate a mix of covalent and noncovalent species in CBCR Ava_3771.[52] Based on this analysis of denatured spectra and of photoconversion of denatured samples, we conclude that both photostates of Anacy_4718g3 contain a covalent PCB adduct despite the fact that 15Z PCB adducts typically absorb at 530-670 nm rather than 720-740 nm.[17, 22, 33, 41, 43, 45, 50, 51, 54, 56, 82, 83] We next characterized Anacy_4718g3 after co-expression with other bilins (Fig. 1). BV did not bind efficiently, but Anacy_4718g3 adducts with phytochromobilin (PB) and phycoerythrobilin (PEB) were obtained (Fig. 6A). Anacy_4718g3-PB exhibited a red shift of the far-red-absorbing state to 752 nm relative to Anacy_4718g3-PCB, but photoconversion with far-red light resulted in formation of a photoproduct with a peak absorption maximum nearly identical that of Anacy_4718g3-PCB (Figs. 6B-C & Table 1). Denaturation analysis confirmed the presence of PB (Fig. 6D). The 18-ethyl moiety of PCB is instead an 18-vinyl in PB (Fig. 1), providing one more double bond in the conjugated π-electron system of PB. Hence, PB adducts of biliproteins are usually red-shifted relative to PCB adducts.[22, 46, 50, 56, 84] The red shift observed for Anacy_4718g3-PB relative to Anacy_4718g3-PCB is consistent with those observed for a broad range of CBCRs upon introduction of the 18-vinyl moiety (Fig. 7A). By contrast, the extinction coefficient of the PCB adduct did not follow the general correlation between peak wavelength and extinction coefficient observed for PCB and phycoviolobilin adducts of most other CBCRs (Fig. 7B & Table S3).


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Biochemistry

PEB differs from PCB and PB in having a saturated 15,16-bond that results in loss of conjugation from the bilin D-ring (Fig. 1). PEB adducts of phytochromes are therefore blue-shifted and cannot undergo photoconversion.[20] Anacy_4718g3-PEB exhibited peak absorption at 610 nm (Fig. 6A), blue-shifted relative to the PCB adduct, and was photoinactive (Fig. 6C). However, the absorption maximum of Anacy4718g3-PEB is redshifted ca. 50 nm relative to PEB adducts of other CBCRs (550-560 nm).[49] Denaturation analysis confirmed the presence of PEB (Fig. 6E). A linear relationship was observed between the peak wavelengths of native and denatured Anacy_4718g3 assembled with PEB, PCB, or PB chromophores for the 15Z configuration of PCB and PB (Fig. 7C). This correlation implies that spectral tuning of all three chromophores by Anacy_4718g3 is comparable, despite the lack of a conjugated D-ring in the PEB adduct. Taken together, these results establish the C15 methine bridge between the C- and Drings as the site of primary photochemistry and establish the A-, B-, and C-ring conjugated system as the site of the pronounced red shift of the 15Z states of bilin adducts of Anacy_4718g3. Fluorescence properties of far-red CBCRs. The peak absorption observed for far-red CBCRs is well into the far-red/near-IR window of maximum penetrance in animal tissues.[31] The only biliproteins known to absorb at longer wavelengths are BVcontaining bacteriophytochromes in the 15E Pfr state.[80] Unfortunately, known phytochromes exhibit little to no fluorescence from the Pfr state, with extremely shortlived excited states.[85-87] We reasoned that the 15Z far-red states of these newly characterized CBCRs might exhibit higher near-IR fluorescence, because the bilin chromophore adopts the 15Z configuration also found in phytochromes engineered for


Biochemistry

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higher fluorescence quantum yield.[21-24, 88] We chose to focus on far-red/orange CBCRs because the two photostates in such proteins have greater spectral separation. All three far-red/orange CBCRs were characterized by fluorescence spectroscopy. These measurements were complicated by the presence of porphyrin (Fig. 4A, asterisk), and fluorescence from the far-red state of Anacy_4718g3 could not be detected against this background (Fig. 8A). Far-red and near-infrared fluorescence could be detected for both Anacy_2551g3 and Cyan7822_4053g2 (Fig. 8B-C). Interestingly, the excitation spectrum of both proteins contained multiple peaks (Fig. 8B-C) that we ascribe to heterogeneity of the far-red state. Fluorescence emission of Anacy_2551g3 extended well into the near-IR (Fig. 9A-B). The observed Stokes shift for the far-red/near-IR state was small, with fluorescence emission peaking at approximately 740 nm for both Anacy_2551g3 and Cyan7822_4053g2. This Stokes shift followed the general trend seen for other CBCRs (Fig. 8D). The fluorescence quantum yield for Anacy_2551g3 was estimated at 1.2% using the ratio method,[22] plotting integrated emission versus absorbance for a dilution series with Alexa 750 as standard (Fig. 9C). These results establish far-red CBCRs as fluorescent, with some examples exhibiting modest near-IR fluorescence detectable at very long wavelengths. Discussion Our work reveals two conserved branches of far-red CBCRs. Three representative proteins exhibiting similar far-red/orange photocycles belong to the first branch. Two of these exhibited detectable near-infrared fluorescence from the far-red-absorbing state. Two representative proteins exhibiting similar far-red/red photocycles belong to the


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second branch. Denaturation analysis demonstrated that far-red sensing at 728-740 nm utilizes a covalent 15Z PCB chromophore similar to that found in other CBCRs and in cyanobacterial and algal phytochromes.[41, 45, 46, 50, 53, 56, 82, 89] Remarkably, this indicates that the same chromophore precursor provides CBCRs with the ability to detect light ranging from 330 to 740 nm through diverse tuning mechanisms. CBCR tuning mechanisms characterized to date provide strategies for blue shifting peak absorption relative to a protonated, cationic bilin π system.[17, 42, 46, 71, 73] The extreme red shift reported here therefore implies the existence of a previously unrecognized tuning mechanism. Although there are obvious parallels between far-red CBCRs and the far-red-absorbing Pfr states of phytochromes, there are also striking differences. It is therefore unclear whether the mechanisms responsible for far-red peak absorption in phytochromes and CBCRs are the same. These novel CBCRs absorb far-red light in the 15Z chromophore configuration rather than the 15E configuration of the phytochrome Pfr state.[90, 91] Most phytochromes require both the bilin-binding GAF domain and the adjacent PHY domain for Pfr formation,[22, 32, 92, 93] whereas far-red CBCRs lack PHY domains altogether (Fig. S8). Previous studies indicate that different phytochromes generate the Pfr state in different ways,[66] and there is no generally accepted model explaining far-red absorption of PCB or PB in the phytochrome Pfr state.[94] It is known that red-shifted 15Z bilin species can be observed as noncovalent adducts in phycobiliproteins,[95] but we have demonstrated that the chromophore is covalently attached in these newly described far-red CBCRs. A variety of models for the 15Z far-red-absorbing state in these proteins must thus be considered.


Biochemistry

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Denaturation analysis confirms that far-red CBCRs employ 15,16-photoisomerization as in other CBCRs,[36, 38, 59, 60] establishing D-ring rotation as the site of photochemistry. The photochemically inactive PEB adduct of Anacy4718g3 also exhibits a red shift comparable to those seen for the other two bilin adducts (Fig. 7C). The D-ring in PEB is not conjugated with the rest of the chromophore (Fig. 1), so the red shift of the PEB adduct must be due to protein-dependent perturbations of the conjugated ABC-ring system. By contrast, the red shift of the PB adduct of Anacy4718g3 relative to that of its PCB adduct is comparable to those seen in other CBCRs (Fig. 7A). Therefore, the tuning mechanism generating far-red absorption in Anacy_4718g3 and related proteins is independent of the double bond between the C- and D-ring. Far-red CBCRs could use a tuning mechanism that does not occur in phytochromes. For example, the far-red state could arise due to formation of the lactim tautomer at the Aring, consistent with the known red shift of O-alkylated bilins but inconsistent with the known protonation state of Pfr phytochrome.[90, 96, 97] Anionic bilin π systems also exhibit substantial red shifts[98, 99] but are again incompatible with the phytochrome Pfr protonation state. The presence of multiple conserved Trp residues in far-red/orange CBCRs (Fig. S1) is consistent with the role of Trp residues in red-shifting phycobiliprotein chromophores,[100] although the red shift observed in phycobiliproteins is much smaller than that observed in this work. The presence of Trp residues proximal to the chromophore also raises the possibility of a previously unknown charge-transfer process generating a labile species not observed in the denaturation assay, a situation somewhat analogous to charge-transfer processes in the blue light receptor cryptochrome.[101, 102] Any of these mechanisms could explain the anomalously low


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extinction coefficients observed for far-red states described in this study (Fig. 7B), because the resulting chromophore structures would chemically differ from the protonated PCB and phycoviolobilin chromophores that establish the observed trend. Consistent with this point, the other exception for the general correlation between extinction coefficient and peak wavelength observed in 15Z PCB adducts is greenabsorbing states such as that of RcaE, which is known to have a deprotonated bilin ring system[17] and hence is distinct from the protonated ring systems that exhibit this correlation. Elucidating the basis for far-red sensing in these proteins will thus require further studies. Our characterization of both far-red/orange and far-red/red CBCRs implies that the farred/orange branch must have a distinct mechanism for spectral tuning of the orangeabsorbing 15E photoproduct. The orange-absorbing photoproducts observed in Anacy_4718g3, Anacy_2551g3, and Cyan7822_4053g2 are very similar, exhibiting a slight blue shift relative to denatured 15E PCB adducts as well as a characteristic lineshape (Fig. 3). Moreover, no red shift is observed in the Anacy_4718g3-PB photoproduct relative to the Anacy_4718g3-PCB photoproduct (Table 1). The absence of such a PB blue shift has also been observed in the teal-DXCF CBCR lineage, in which the photoproduct D-ring is trapped in a twisted geometry reducing conjugation to yield a blue-shifted chromophore with a similar lineshape.[71] It is thus possible that a similar trapped-twist mechanism acts to tune the orange-absorbing photoproduct. To examine this hypothesis, we plotted 15E photoproduct blue shift versus 15Z peak wavelength for a range of trapped-twist CBCRs, for a range of CBCRs lacking residues required for trapped-twist photoproducts and hence having „relaxed‟ photoproducts, and for denatured


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samples.[50, 52, 71] CBCRs with trapped-twist photoproducts exhibited a linear relationship between these spectral parameters (Fig. 7D), and far-red/orange CBCRs followed this correlation. CBCRs with relaxed photoproducts were more similar to denatured samples, and both far-red/red CBCRs examined in this study followed the relaxed trend (Fig. 7D). This analysis implicates some type of trapped-twist tuning in the orange-absorbing photoproducts of far-red CBCRs. Interestingly, comparison of the Aspmotifs of far-red/red CBCRs to those of far-red/orange CBCRs reveals that far-red/red CBCRs lack one of the Trp residues found in far-red/orange CBCRs (Fig. S1). This Trp residue may thus constrain chromophore motions during photoconversion. Aromatic residues are similarly implicated in spectral tuning in other CBCR lineages,[52, 71, 103] and recent work also implicates Trp residues in spectral tuning of phycobiliproteins.[100] It is thus possible that a similar effect provides spectral tuning in far-red/orange CBCRs. Our work also implicates additional unknown far-red to near-IR photobiology in cyanobacteria. The newly recognized far-red CBCRs do not correlate with known far-red photobiological responses. For example, the filamentous cyanobacterium Leptolyngbya sp. JSC-1 is known to exhibit far-red light-induced photoacclimation (FaRLiP), but this organism lacks a far-red CBCR (Tables S1-S2) and FaRLiP is controlled by a different photosensor.[93,

104-106]

The

more

recently

described

low-light-induced

photoacclimation (LoLiP) response also does not correlate with FR CBCRs, as shown by the presence of a far-red CBCR (Table S1) but absence of LoLiP in Leptolyngbya sp. PCC 7104.[105] Far-red CBCRs are present in both unicellular and filamentous cyanobacteria and are associated with multiple histidine kinase lineages and domain architectures (Fig. 2 & Tables S1-S2). By contrast, the known CCA regulator RcaE is


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part of coherent phylogenetic clusters in both the CBCR and histidine kinase trees (Fig. 2, shaded green box). This RcaE cluster correlates well with the presence of type III CCA in the host organisms (Table S4). Far-red CBCRs are therefore likely to be associated with multiple physiological responses. Finally, our work also raises the possibility of using far-red CBCRs „lead compounds‟ to develop fluorescent reporters, optical or photoacoustic contrast agents, or synthetic biology reagents responding to far-red or near-infrared light. Far-red CBCRs exhibit a unique combination of small size, far-red peak absorption, and detectable near-infrared fluorescence not found in other phytochromes or CBCRs (Table 2). Protein engineering has allowed development of a range of phytochrome variants with peak fluorescence at ≤730 nm (Table 2). Interestingly, variants with peak fluorescence at >700 nm have fluorescence quantum yields of 4-8% after engineering and can be used in imaging applications.[24, 25, 107, 108] Anacy_2551g3 thus is a promising lead compound for development of similar reagents, given its quantum yield of 1.2% and peak emission at 740 nm prior to any engineering. Far-red CBCRs thus hold great promise as fluorescent reporters and optical or photoacoustic contrast agents in systems for which the far-red/near-IR window is critical for optimal performance. [21, 23-25]It should also be possible to couple far-red CBCRs to alternative outputs for modulating various aspects of eukaryotic biology, as has been done for bacteriophytochromes.[29, 30] Far-red CBCRs thus extend a series of studies[39, 41, 44-48, 50-53, 109] establishing CBCRs as having the broadest light sensing range of known photoreceptor families. As modular light sensors ranging from


Biochemistry

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ca. 330 nm to 750 nm, CBCRs span the full range of the electromagnetic spectrum amenable to oxygenic photosynthesis by cyanobacteria.

Acknowledgements We thank Elsie Campbell and Jack Meeks for the gift of genomic DNA and Rei Narikawa and Yuu Hirose for helpful discussions. This work was supported by a grant from the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, United States Department of Energy (DOE DESC0002395 to J.C.L.).


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Biochemistry

Table 1: Spectral properties of CBCRs in this study1

Protein

Construct

Bilin

15Z max

15E max

SAR

Anacy_4718g3

intein-CBD

PCB

740

590

0.16

Anacy_4718g3

intein-CBD

PB

752

590

0.12

Anacy_4718g3

intein-CBD

PEB

6102

–

0.37

Anacy_4718g3

intein-CBD

BV

–

–

500

663

676

0.005

Cph1 Y176H

PCB

>500

645

671

0.145

Cph1 Y176H

PB

>500

655

683

0.067

IFP1.4

BV

ca. 300

684

708

0.07

IFP2.0

BV

ca. 300

690

711

0.08

mIFP

BV

ca. 300

683

704

0.08

Wi-PHY

BV

ca. 300

701

719

0.047

iRFP/iRFP713

BV

ca. 300

690

713

0.063

iRFP720

BV

ca. 300

702

720

0.060

GAF-FP

PCB

Identification of Cyanobacteriochromes Detecting ... - ACS Publications

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