Identification of Cyanobacteriochromes Detecting Far-Red Light

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 far-red 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 (λ_max = 725–755 nm)/orange (λ_max = 590–600 nm) and far-red/red (λ_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 far-red/orange CBCRs evolved after a complex transition from green/red CBCRs known for regulating complementary chromatic acclimation. Incorporation of different bilin chromophores demonstrated 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 region. 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

Light-Regulated Synthesis of Cyclic-di-GMP by a Bidomain Construct of the Cyanobacteriochrome Tlr0924 (SesA) without Stable Dimerization

Phytochromes and cyanobacteriochromes (CBCRs) use double-bond photoisomerization of their linear tetrapyrrole (bilin) chromophores within cGMP-specific phosphodiesterases/adenylyl cyclases/FhlA (GAF) domain-containing photosensory modules to regulate activity of C-terminal output domains. CBCRs exhibit photocycles that are much more diverse than those of phytochromes and are often found in large modular proteins such as Tlr0924 (SesA), one of three blue light regulators of cell aggregation in the cyanobacterium <i>Thermosynechococcus elongatus</i>. Tlr0924 contains a single bilin-binding GAF domain adjacent to a C-terminal diguanylate cyclase (GGDEF) domain whose catalytic activity requires formation of a dimeric transition state presumably supported by a multidomain extension at its N-terminus. To probe the structural basis of light-mediated signal propagation from the photosensory input domain to a signaling output domain for a representative CBCR, these studies explore the properties of a bidomain GAF–GGDEF construct of Tlr0924 (Tlr0924Δ) that retains light-regulated diguanylate cyclase activity. Surprisingly, circular dichroism spectroscopy and size exclusion chromatography data do not support formation of stable dimers in either the blue-absorbing ^15<i>Z</i>P_b dark state or the green-absorbing ^15<i>E</i>P_g photoproduct state of Tlr0924Δ. Analysis of variants containing site-specific mutations reveals that proper signal transmission requires both chromophorylation of the GAF domain and individual residues within the amphipathic linker region between GAF and GGDEF domains. On the basis of these data, we propose a model in which bilin binding and light signals are propagated from the GAF domain via the linker to alter the equilibrium and interconversion dynamics between active and inactive conformations of the GGDEF domain to favor or disfavor formation of catalytically competent dimers

Mechanistic Insight into the Photosensory Versatility of DXCF Cyanobacteriochromes

Cyanobacteriochromes (CBCRs) are photosensory proteins related to the red/far-red phytochromes. Like phytochromes, CBCRs use linear tetrapyrrole (bilin) chromophores covalently attached via a thioether linkage to a conserved Cys residue also found in plant and cyanobacterial phytochromes. Unlike almost all phytochromes, CBCRs require only an isolated GAF domain to undergo efficient, reversible photocycles that are responsible for their broad light sensing range, spanning the visible to the near ultraviolet (UV). Sensing of blue, violet, and near-UV light by CBCRs requires another Cys residue proposed to form a second linkage to the bilin precursor. Light triggers 15,16-double bond isomerization as in phytochromes. After photoisomerization, elimination of the second linkage frequently occurs, thus yielding a large red shift of the stable photoproducts. Here we examine this process for representative DXCF CBCRs, a large subfamily named for the conserved Asp-Xaa-Cys-Phe motif that contains their second Cys residue. DXCF CBCRs with such dual-Cys photocycles yield a wide diversity of photoproducts absorbing teal, green, or orange light. Using a combination of CD spectroscopy, chemical modification, and bilin substitution experiments with recombinant CBCRs from <i>Thermosynechococcus elongatus</i> and <i>Nostoc punctiforme</i> expressed in <i>Escherichia coli</i>, we establish that second-linkage elimination is required for all of these photocycles. We also identify deconjugation of the D-ring as the mechanism for specific detection of teal light, at approximately 500 nm. Our studies thus provide new mechanistic insight into the photosensory versatility of this important family of photosensory proteins

Red/Green Cyanobacteriochromes: Sensors of Color and Power

Phytochromes are red/far-red photoreceptors using cysteine-linked linear tetrapyrrole (bilin) chromophores to regulate biological responses to light. Light absorption triggers photoisomerization of the bilin between the 15<i>Z</i> and 15<i>E</i> photostates. The related cyanobacteriochromes (CBCRs) extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. Several subfamilies of CBCRs have been described. Representatives of one such subfamily, including AnPixJ and NpR6012g4, exhibit red/green photocycles in which the 15<i>Z</i> photostate is red-absorbing like that of phytochrome but the 15<i>E</i> photoproduct is instead green-absorbing. Using recombinant expression of individual CBCR domains in <i>Escherichia coli</i>, we fully survey the red/green subfamily from the cyanobacterium <i>Nostoc punctiforme</i>. In addition to 14 new photoswitching CBCRs, one apparently photochemically inactive protein exhibiting intense red fluorescence was observed. We describe a novel orange/green photocycle in one of these CBCRs, NpF2164g7. Dark reversion varied in this panel of CBCRs; some examples were stable as the 15<i>E</i> photoproduct for days, while others reverted to the 15<i>Z</i> dark state in minutes or even seconds. In the case of NpF2164g7, dark reversion was so rapid that reverse photoconversion of the green-absorbing photoproduct was not significant in restoring the dark state, resulting in a broadband response to light. Our results demonstrate that red/green CBCRs can thus act as sensors for the color or intensity of the ambient light environment

Conserved Phenylalanine Residues Are Required for Blue-Shifting of Cyanobacteriochrome Photoproducts

Cyanobacteriochromes (CBCRs) are cyanobacterial photosensory proteins distantly related to phytochromes. Both phytochromes and CBCRs reversibly convert between dark-stable and photoproduct states upon photoisomerization of their linear tetrapyrrole (bilin) chromophores. While most phytochromes convert between a red-absorbing dark state and a far-red-absorbing photoproduct, CBCRs exhibit spectral responses spanning the entire near-ultraviolet and visible spectrum. For example, red/green CBCRs such as AnPixJ and NpR6012g4 exhibit a red-absorbing dark state similar to that of phytochrome, but photoconversion yields a green-absorbing photoproduct. “Teal-DXCF” CBCRs convert from blue- or green-absorbing dark states to yield photoproducts with very narrow absorption in the teal region of the spectrum (approximately 500 nm). The recent determination of a crystal structure of AnPixJ in its red-absorbing dark state led to the proposal that movement of a Trp residue (the “lid Trp”) upon photoconversion would allow solvation of the photoproduct, thereby producing a large blue-shift. We find that substitution of the lid Trp has little effect on the NpR6012g4 photoproduct. Instead, two Phe residues conserved in red/green and teal-DXCF CBCRs are essential for determining photoproduct absorption in both CBCR groups with no significant influence on the dark-adapted state. We propose that these Phe residues constrain chromophore movement after primary photoisomerization. This work supports a trapped–twist mechanism for generating both red/green and teal-DXCF photoproducts

Characterization of Red/Green Cyanobacteriochrome NpR6012g4 by Solution Nuclear Magnetic Resonance Spectroscopy: A Hydrophobic Pocket for the C15-<i>E,anti</i> Chromophore in the Photoproduct

Cyanobacteriochromes (CBCRs) are cyanobacterial photosensory proteins distantly related to phytochromes. Like phytochromes, CBCRs reversibly photoconvert between a dark-stable state and a photoproduct via photoisomerization of the 15,16-double bond of their linear tetrapyrrole (bilin) chromophores. CBCRs provide cyanobacteria with complete coverage of the visible spectrum and near-ultraviolet region. One CBCR subfamily, the canonical red/green CBCRs typified by AnPixJg2 and NpR6012g4, can function as sensors of light color or intensity because of their great variation in photoproduct stability. The mechanistic basis for detection of green light by the photoproduct state in this subfamily has proven to be a challenging research topic, with competing hydration and trapped-twist models proposed. Here, we use ¹³C-edited and ¹⁵N-edited ¹H–¹H NOESY solution nuclear magnetic resonance spectroscopy to probe changes in chromophore configuration and protein–chromophore interactions in the NpR6012g4 photocycle. Our results confirm a C15-<i>Z</i>,<i>anti</i> configuration for the red-absorbing dark state and reveal a C15-<i>E</i>,<i>anti</i> configuration for the green-absorbing photoproduct. The photoactive chromophore D-ring is located in a hydrophobic environment in the photoproduct, surrounded by both aliphatic and aromatic residues. Characterization of variant proteins demonstrates that no aliphatic residue is essential for photoproduct tuning. Taken together, our results support the trapped-twist model over the hydration model for the red/green photocycle of NpR6012g4

Characterization of Red/Green Cyanobacteriochrome NpR6012g4 by Solution Nuclear Magnetic Resonance Spectroscopy: A Protonated Bilin Ring System in Both Photostates

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to phytochromes. Both CBCRs and phytochromes use photoisomerization of a linear tetrapyrrole (bilin) chromophore to photoconvert between two states with distinct spectral and biochemical properties, the dark state and the photoproduct. The isolated CBCR domain NpR6012g4 from <i>Nostoc punctiforme</i> is a well-characterized member of the canonical red/green CBCR subfamily, photosensory domains that can function as sensors for light color or intensity to regulate phototactic responses of filamentous cyanobacteria. Such red/green CBCRs utilize conserved Phe residues to tune the photoproduct for green light absorption, but conflicting interpretations of the photoproduct chromophore structure have been proposed. In the hydration model, the proposed photoproduct state is extensively solvated, with a loosely bound, conformationally flexible chromophore. In the trapped-twist model, the photoproduct chromophore is sterically constrained by hydrophobic amino acids, including the known Phe residues. Here, we have characterized chromophore structure in NpR6012g4 using solution nuclear magnetic resonance spectroscopy and a series of labeled chromophores. Four NH resonances are assigned for both the red-absorbing dark state and the green-absorbing photoproduct. Moreover, observed ¹³C chemical shifts are in good agreement with those obtained for protonated rather than deprotonated bilins in <i>ab initio</i> calculations. Our results demonstrate that NpR6012g4 has a protonated, cationic bilin π system in both photostates, consistent with a photoproduct structure in which the chromophore is not extensively hydrated

Femtosecond Photodynamics of the Red/Green Cyanobacteriochrome NpR6012g4 from <i>Nostoc punctiforme</i>. 2. Reverse Dynamics

Phytochromes are red/far-red photosensory proteins that utilize photoisomerization of a linear tetrapyrrole (bilin) chromophore to photoconvert reversibly between red- and far-red-absorbing forms (P_r and P_fr, respectively). Cyanobacteriochromes (CBCRs) are related photosensory proteins with more diverse spectral sensitivity. The mechanisms that underlie this spectral diversity have not yet been fully elucidated. One of the main CBCR subfamilies photoconverts between a red-absorbing 15<i>Z</i> ground state, like the familiar P_r state of phytochromes, and a green-absorbing photoproduct (^15<i>E</i>P_g). We have previously used the red/green CBCR NpR6012g4 from the cyanobacterium <i>Nostoc punctiforme</i> to examine ultrafast photodynamics of the forward photoreaction. Here, we examine the reverse reaction. Using excitation-interleaved transient absorption spectroscopy with broadband detection and multicomponent global analysis, we observed multiphasic excited-state dynamics. Interleaved excitation allowed us to identify wavelength-dependent shifts in the ground-state bleach that equilibrated on a 200 ps time scale, indicating ground-state heterogeneity. Compared to the previously studied forward reaction, the reverse reaction has much faster excited-state decay time constants and significantly higher photoproduct yield. This work thus demonstrates striking differences between the forward and reverse reactions of NpR6012g4 and provides clear evidence of ground-state heterogeneity in the phytochrome superfamily

Reactive Ground-State Pathways Are Not Ubiquitous in Red/Green Cyanobacteriochromes

Recent characterization of the red/green cyanobacteriochrome (CBCR) NpR6012g4 revealed a high quantum yield for its forward photoreaction [J. Am. Chem. Soc. 2012, 134, 130−133] that was ascribed to the activity of hidden, productive ground-state intermediates. The dynamics of the pathways involving these ground-state intermediates was resolved with femtosecond dispersed pump–dump–probe spectroscopy, the first such study reported for any CBCR. To address the ubiquity of such second-chance initiation dynamics (SCID) in CBCRs, we examined the closely related red/green CBCR NpF2164g6 from <i>Nostoc punctiforme</i>. Both NpF2164g6 and NpR6012g4 use phycocyanobilin as the chromophore precursor and exhibit similar excited-state dynamics. However, NpF2164g6 exhibits a lower quantum yield of 32% for the generation of the isomerized Lumi-R primary photoproduct, compared to 40% for NpR6012g4. This difference arises from significantly different ground-state dynamics between the two proteins, with the SCID mechanism deactivated in NpF2164g6. We present an integrated inhomogeneous target model that self-consistently fits the pump–probe and pump–dump–probe signals for both forward and reverse photoreactions in both proteins. This work demonstrates that reactive ground-state intermediates are not ubiquitous phenomena in CBCRs

Protonation Heterogeneity Modulates the Ultrafast Photocycle Initiation Dynamics of Phytochrome Cph1

Phytochrome proteins utilize ultrafast photoisomerization of a linear tetrapyrrole chromophore to detect the ratio of red to far-red light. Femtosecond photodynamics in the PAS-GAF-PHY photosensory core of the Cph1 phytochrome from <i>Synechocystis</i> sp. PCC6803 (Cph1Δ) were resolved with a dual-excitation-wavelength-interleaved pump–probe (DEWI) approach with two excitation wavelengths (600 and 660 nm) at three pH values (6.5, 8.0, and 9.0). Observed spectral and kinetic heterogeneity in the excited-state dynamics were described with a self-consistent model comprised of three spectrally distinct populations with different protonation states (P_r-I, P_r-II, and P_r-III), each composed of multiple kinetically distinct subpopulations. Apparent partitioning among these populations is dictated by pH, temperature, and excitation wavelength. Our studies provide insight into photocycle initiation dynamics at physiological temperatures, implicate the low-pH/low-temperature P_r-I state as the photoactive state <i>in vitro</i>, and implicate an internal hydrogen-bonding network in regulating the photochemical quantum yield