Showing 1 - 25 of 28 results
Signaling, Deconstructed: Using Optogenetics to Dissect and Direct Information Flow in Biological Systems.
Cells receive enormous amounts of information from their environment. How they act on this information-by migrating, expressing genes, or relaying signals to other cells-comprises much of the regulatory and self-organizational complexity found across biology. The "parts list" involved in cell signaling is generally well established, but how do these parts work together to decode signals and produce appropriate responses? This fundamental question is increasingly being addressed with optogenetic tools: light-sensitive proteins that enable biologists to manipulate the interaction, localization, and activity state of proteins with high spatial and temporal precision. In this review, we summarize how optogenetics is being used in the pursuit of an answer to this question, outlining the current suite of optogenetic tools available to the researcher and calling attention to studies that increase our understanding of and improve our ability to engineer biology. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 23 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Synthetic Biological Approaches for Optogenetics and Tools for Transcriptional Light‐Control in Bacteria.
Light has become established as a tool not only to visualize and investigate but also to steer biological systems. This review starts by discussing the unique features that make light such an effective control input in biology. It then gives an overview of how light‐control came to progress, starting with photoactivatable compounds and leading up to current genetic implementations using optogenetic approaches. The review then zooms in on optogenetics, focusing on photosensitive proteins, which form the basis for optogenetic engineering using synthetic biological approaches. As the regulation of transcription provides a highly versatile means for steering diverse biological functions, the focus of this review then shifts to transcriptional light regulators, which are presented in the biotechnologically highly relevant model organism Escherichia coli.
Dual Systems for Enhancing Control of Protein Activity through Induced Dimerization Approaches.
To reveal the underpinnings of complex biological systems, a variety of approaches have been developed that allow switchable control of protein function. One powerful approach for switchable control is the use of inducible dimerization systems, which can be configured to control activity of a target protein upon induced dimerization triggered by chemicals or light. Individually, many inducible dimerization systems suffer from pre‐defined dynamic ranges and overwhelming sensitivity to expression level and cellular context. Such systems often require extensive engineering efforts to overcome issues of background leakiness and restricted dynamic range. To address these limitations, recent tool development efforts have explored overlaying dimerizer systems with a second layer of regulation. Albeit more complex, the resulting layered systems have enhanced functionality, such as tighter control that can improve portability of these tools across platforms.
Steering Molecular Activity with Optogenetics: Recent Advances and Perspectives.
Optogenetics utilizes photosensitive proteins to manipulate the localization and interaction of molecules in living cells. Because light can be rapidly switched and conveniently confined to the sub‐micrometer scale, optogenetics allows for controlling cellular events with an unprecedented resolution in time and space. The past decade has witnessed an enormous progress in the field of optogenetics within the biological sciences. The ever‐increasing amount of optogenetic tools, however, can overwhelm the selection of appropriate optogenetic strategies. Considering that each optogenetic tool may have a distinct mode of action, a comparative analysis of the current optogenetic toolbox can promote the further use of optogenetics, especially by researchers new to this field. This review provides such a compilation that highlights the spatiotemporal accuracy of current optogenetic systems. Recent advances of optogenetics in live cells and animal models are summarized, the emerging work that interlinks optogenetics with other research fields is presented, and exciting clinical and industrial efforts to employ optogenetic strategy toward disease intervention are reported.
Photoactivated Adenylyl Cyclases: Fundamental Properties and Applications.
Photoactivated adenylyl cyclase (PAC) was first discovered to be a sensor for photoavoidance in the flagellate Euglena gracilis. PAC is a flavoprotein that catalyzes the production of cAMP upon illumination with blue light, which enables us to optogenetically manipulate intracellular cAMP levels in various biological systems. Recent progress in genome sequencing has revealed several related proteins in bacteria and ameboflagellates. Among them, the PACs from sulfur bacterium Beggiatoa sp. and cyanobacterium Oscillatoria acuminata have been well characterized, including their crystalline structure. Although there have not been many reported optogenetic applications of PACs so far, they have the potential to be used in various fields within bioscience.
Improved Photocleavable Proteins with Faster and More Efficient Dissociation.
The photocleavable protein (PhoCl) is a green-to-red photoconvertible fluorescent protein that, when illuminated with violet light, undergoes main chain cleavage followed by spontaneous dissociation of the resulting fragments. The first generation PhoCl (PhoCl1) exhibited a relative slow rate of dissociation, potentially limiting its utilities for optogenetic control of cell physiology. In this work, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast ratio and PhoCl2f with faster dissociation. We characterized the performance of these new variants as purified proteins and expressed in cultured cells. Our results demonstrate that PhoCl2 variants exhibit faster and more efficient dissociation, which should enable improved optogenetic manipulations of protein localization and protein-protein interactions in living cells.
A light way for nuclear cell biologists.
The nucleus is a very complex organelle present in eukaryotic cells. Having the crucial task to safeguard, organize and manage the genetic information, it must tightly control its molecular constituents, its shape and its internal architecture at any given time. Despite our vast knowledge of nuclear cell biology, much is yet to be unraveled. For instance, only recently we came to appreciate the existence of a dynamic nuclear cytoskeleton made of actin filaments that regulates processes such as gene expression, DNA repair and nuclear expansion. This suggests further exciting discoveries ahead of us. Modern cell biologists embrace a new methodology relying on precise perturbations of cellular processes that require a reversible, highly spatially-confinable, rapid, inexpensive and tunable external stimulus: light. In this review, we discuss how optogenetics, the state-of-the-art technology that uses genetically-encoded light-sensitive proteins to steer biological processes, can be adopted to specifically investigate nuclear cell biology.
Optogenetics and biosensors set the stage for metabolic cybergenetics.
Cybergenetic systems use computer interfaces to enable feed-back controls over biological processes in real time. The complex and dynamic nature of cellular metabolism makes cybergenetics attractive for controlling engineered metabolic pathways in microbial fermentations. Cybergenetics would not only create new avenues of research into cellular metabolism, it would also enable unprecedented strategies for pathway optimization and bioreactor operation and automation. Implementation of metabolic cybergenetics, however, will require new capabilities from actuators, biosensors, and control algorithms. The recent application of optogenetics in metabolic engineering, the expanding role of genetically encoded biosensors in strain development, and continued progress in control algorithms for biological processes suggest that this technology will become available in the not so distant future.
SPLIT: Stable Protein Coacervation using a Light Induced Transition.
Protein coacervates serve as hubs to concentrate and sequester proteins and nucleotides and thus function as membrane-less organelles to manipulate cell physiology. We have engineered a coacervating protein to create tunable, synthetic membrane-less organelles that assemble in response to a single pulse of light. Coacervation is driven by the intrinsically disordered RGG domain from the protein LAF-1, and opto-responsiveness is coded by the protein PhoCl which cleaves in response to 405 nm light. We developed a fusion protein containing a solubilizing maltose binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellular-sized water-in-oil emulsions. An optimized version of this system displayed light-induced coacervation in Saccharomyces cerevisiae. The methods described here provide novel strategies for inducing protein phase separation using light.
Recent advances in the use of genetically encodable optical tools to elicit and monitor signaling events.
Cells rely on a complex network of spatiotemporally regulated signaling activities to effectively transduce information from extracellular cues to intracellular machinery. To probe this activity architecture, researchers have developed an extensive molecular tool kit of fluorescent biosensors and optogenetic actuators capable of monitoring and manipulating various signaling activities with high spatiotemporal precision. The goal of this review is to provide readers with an overview of basic concepts and recent advances in the development and application of genetically encodable biosensors and optogenetic tools for understanding signaling activity.
Hydrogels With Tunable Mechanical Properties Based on Photocleavable Proteins.
Hydrogels with photo-responsive mechanical properties have found broad biomedical applications, including delivering bioactive molecules, cell culture, biosensing, and tissue engineering. Here, using a photocleavable protein, PhoCl, as the crosslinker we engineer two types of poly(ethylene glycol) hydrogels whose mechanical stability can be weakened or strengthened, respectively, upon visible light illumination. In the photo weakening hydrogels, photocleavage leads to rupture of the protein crosslinkers, and decrease of the mechanical properties of the hydrogels. In contrast, in the photo strengthening hydrogels, by properly choosing the crosslinking positions, photocleavage does not rupture the crosslinking sites but exposes additional cryptical reactive cysteine residues. When reacting with extra maleimide groups in the hydrogel network, the mechanical properties of the hydrogels can be enhanced upon light illumination. Our study indicates that photocleavable proteins could provide more designing possibilities than the small-molecule counterparts. A proof-of-principle demonstration of spatially controlling the mechanical properties of hydrogels was also provided.
Functional Modulation of Receptor Proteins on Cellular Interface with Optogenetic System.
In multicellular organisms, living cells cooperate with each other to exert coordinated complex functions by responding to extracellular chemical or physical stimuli via proteins on the plasma membrane. Conventionally, chemical signal transduction or mechano-transduction has been investigated by chemical, genetic, or physical perturbation; however, these methods cannot manipulate biomolecular reactions at high spatiotemporal resolution. In contrast, recent advances in optogenetic perturbation approaches have succeeded in controlling signal transduction with external light. The methods have enabled spatiotemporal perturbation of the signaling, providing functional roles of the specific proteins. In this chapter, we summarize recent advances in the optogenetic tools that modulate the function of a receptor protein. While most optogenetic systems have been devised for controlling ion channel conductivities, the present review focuses on the other membrane proteins involved in chemical transduction or mechano-transduction. We describe the properties of natural or artificial photoreceptor proteins used in optogenetic systems. Then, we discuss the strategies for controlling the receptor protein functions by external light. Future prospects of optogenetic tool development are discussed.
Phytochromes and Cyanobacteriochromes: Photoreceptor Molecules Incorporating a Linear Tetrapyrrole Chromophore.
In this chapter, we summarize the molecular mechanisms of the linear tetrapyrrole-binding photoreceptors, phytochromes, and cyanobacteriochromes. We especially focus on the color-tuning mechanisms and conformational changes during the photoconversion process. Furthermore, we introduce current status of development of the optogenetic tools based on these molecules. Huge repertoire of these photoreceptors with diverse spectral properties would contribute to development of multiplex optogenetic regulation. Among them, the photoreceptors incorporating the biliverdin IXα chromophore is advantageous for in vivo optogenetics because this is intrinsic in the mammalian cells, and absorbs far-red light penetrating into deep mammalian tissues.
Elucidating cyclic AMP signaling in subcellular domains with optogenetic tools and fluorescent biosensors.
The second messenger 3',5'-cyclic nucleoside adenosine monophosphate (cAMP) plays a key role in signal transduction across prokaryotes and eukaryotes. Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision. To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited. Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively. In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell. This review provides an overview about optogenetic tools and biosensors to shed light on the subcellular organization of cAMP signaling.
Photocleavable Cadherin Inhibits Cell-to-Cell Mechanotransduction by Light.
Precise integration of individual cell behaviors is indispensable for collective tissue morphogenesis and maintenance of tissue integrity. Organized multicellular behavior is achieved via mechanical coupling of individual cellular contractility, mediated by cell adhesion molecules at the cell-cell interface. Conventionally, gene depletion or laser microsurgery has been used for functional analysis of intercellular mechanotransduction. Nevertheless, these methods are insufficient to investigate either the spatiotemporal dynamics or the biomolecular contribution in cell-cell mechanical coupling within collective multicellular behaviors. Herein, we present our effort in adaption of PhoCl for attenuation of cell-to-cell tension transmission mediated by E-cadherin. To release intercellular contractile tension applied on E-cadherin molecules with external light, a genetically encoded photocleavable module called PhoCl was inserted into the intracellular domain of E-cadherin, thereby creating photocleavable cadherin (PC-cadherin). In response to light illumination, the PC-cadherin cleaved into two fragments inside cells, resulting in attenuating mechanotransduction at intercellular junctions in living epithelial cells. Light-induced perturbation of the intercellular tension balance with surrounding cells changed the cell shape in an epithelial cell sheet. The method is expected to enable optical manipulation of force-mediated cell-to-cell communications in various multicellular behaviors, which contributes to a deeper understanding of embryogenesis and oncogenesis.
Genetically Encoded Photocleavable Linkers for Patterned Protein Release from Biomaterials.
Given the critical role that proteins play in almost all biological processes, there is great interest in controlling their presentation within and release from biomaterials. Despite such outstanding enthusiasm, previously developed strategies in this regard result in ill-defined and heterogeneous populations with substantially decreased activity, precluding their successful application to fragile species including growth factors. Here, we introduce a modular and scalable method for creating monodisperse, genetically encoded chimeras that enable bioactive proteins to be immobilized within and subsequently photoreleased from polymeric hydrogels. Building upon recent developments in chemoenzymatic reactions, bioorthogonal chemistry, and optogenetics, we tether fluorescent proteins, model enzymes, and growth factors site-specifically to gel biomaterials through a photocleavable protein (PhoCl) that undergoes irreversible backbone photoscission upon exposure to cytocompatible visible light (λ ≈ 400 nm) in a dose-dependent manner. Mask-based and laser-scanning lithographic strategies using commonly available light sources are employed to spatiotemporally pattern protein release from hydrogels while retaining their full activity. The photopatterned epidermal growth factor presentation is exploited to promote anisotropic cellular proliferation in 3D. We expect these methods to be broadly useful for applications in diagnostics, drug delivery, and regenerative medicine.
Diverse light responses of cyanobacteria mediated by phytochrome superfamily photoreceptors.
Cyanobacteria are an evolutionarily and ecologically important group of prokaryotes. They exist in diverse habitats, ranging from hot springs and deserts to glaciers and the open ocean. The range of environments that they inhabit can be attributed in part to their ability to sense and respond to changing environmental conditions. As photosynthetic organisms, one of the most crucial parameters for cyanobacteria to monitor is light. Cyanobacteria can sense various wavelengths of light and many possess a range of bilin-binding photoreceptors belonging to the phytochrome superfamily. Vital cellular processes including growth, phototaxis, cell aggregation and photosynthesis are tuned to environmental light conditions by these photoreceptors. In this Review, we examine the physiological responses that are controlled by members of this diverse family of photoreceptors and discuss the signal transduction pathways through which these photoreceptors operate. We highlight specific examples where the activities of multiple photoreceptors function together to fine-tune light responses. We also discuss the potential application of these photosensing systems in optogenetics and synthetic biology.
Programming Bacteria With Light—Sensors and Applications in Synthetic Biology
Photo-receptors are widely present in both prokaryotic and eukaryotic cells, which serves as the foundation of tuning cell behaviors with light. While practices in eukaryotic cells have been relatively established, trials in bacterial cells have only been emerging in the past few years. A number of light sensors have been engineered in bacteria cells and most of them fall into the categories of two-component and one-component systems. Such a sensor toolbox has enabled practices in controlling synthetic circuits at the level of transcription and protein activity which is a major topic in synthetic biology, according to the central dogma. Additionally, engineered light sensors and practices of tuning synthetic circuits have served as a foundation for achieving light based real-time feedback control. Here, we review programming bacteria cells with light, introducing engineered light sensors in bacteria and their applications, including tuning synthetic circuits and achieving feedback controls over microbial cell culture.
Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells.
Class III adenylyl cyclases generate the ubiquitous second messenger cAMP from ATP often in response to environmental or cellular cues. During evolution, soluble adenylyl-cyclase catalytic domains have been repeatedly juxtaposed with signal-input domains to place cAMP synthesis under the control of a wide variety of these environmental and endogenous signals. Adenylyl cyclases with light-sensing domains have proliferated in photosynthetic species depending on light as an energy source, yet are also widespread in non-photosynthetic species. Among such naturally occurring light sensors, several flavin-based photoactivated adenylyl cyclases (PACs) have been adopted as optogenetic tools to manipulate cellular processes with blue light. In this report, we report the discovery of a cyanobacteriochrome-based photoswitchable adenylyl cyclase (cPAC) from the cyanobacterium Microcoleussp. PCC 7113. Unlike flavin-dependent PACs, which must thermally decay to be deactivated, cPAC exhibited a bistable photocycle whose adenylyl cyclase could be reversibly activated and inactivated by blue and green light, respectively. Through domain exchange experiments, we also document the ability to extend the wavelength-sensing specificity of cPAC into the near IR. In summary, our work has uncovered a cyanobacteriochrome-based adenylyl cyclase that holds great potential for design of bistable photoswitchable adenylyl cyclases to fine-tune cAMP-regulated processes in cells. tissues, and whole organisms with light across the visible spectrum and into near IR.
A novel optogenetically tunable frequency modulating oscillator.
Synthetic biology has enabled the creation of biological reconfigurable circuits, which perform multiple functions monopolizing a single biological machine; Such a system can switch between different behaviours in response to environmental cues. Previous work has demonstrated switchable dynamical behaviour employing reconfigurable logic gate genetic networks. Here we describe a computational framework for reconfigurable circuits in E.coli using combinations of logic gates, and also propose the biological implementation. The proposed system is an oscillator that can exhibit tunability of frequency and amplitude of oscillations. Further, the frequency of operation can be changed optogenetically. Insilico analysis revealed that two-component light systems, in response to light within a frequency range, can be used for modulating the frequency of the oscillator or stopping the oscillations altogether. Computational modelling reveals that mixing two colonies of E.coli oscillating at different frequencies generates spatial beat patterns. Further, we show that these oscillations more robustly respond to input perturbations compared to the base oscillator, to which the proposed oscillator is a modification. Compared to the base oscillator, the proposed system shows faster synchronization in a colony of cells for a larger region of the parameter space. Additionally, the proposed oscillator also exhibits lesser synchronization error in the transient period after input perturbations. This provides a strong basis for the construction of synthetic reconfigurable circuits in bacteria and other organisms, which can be scaled up to perform functions in the field of time dependent drug delivery with tunable dosages, and sets the stage for further development of circuits with synchronized population level behaviour.
Distinctive Properties of Dark Reversion Kinetics between Two Red/Green-Type Cyanobacteriochromes and their Application in the Photoregulation of cAMP Synthesis.
Cyanobacteriochromes (CBCRs) are photoreceptors that bind to a linear tetrapyrrole within a conserved cGMP-phosphodiesterase/adenylate cyclase/FhlA (GAF) domain and exhibit reversible photoconversion. Red/green-type CBCR GAF domains that photoconvert between red- (Pr) and green-absorbing (Pg) forms occur widely in various cyanobacteria. A putative phototaxis regulator, AnPixJ, contains multiple red/green-type CBCR GAF domains. We previously reported that AnPixJ's second domain (AnPixJg2) but not its fourth domain (AnPixJg4) shows red/green reversible photoconversion. Herein, we found that AnPixJg4 showed Pr-to-Pg photoconversion and rapid Pg-to-Pr dark reversion, whereas AnPixJg2 showed a barely detectable dark reversion. Site-directed mutagenesis revealed the involvement of six residues in Pg stability. Replacement at the Leu294/Ile660 positions of AnPixJg2/AnPixJg4 showed the highest influence on dark reversion kinetics. AnPixJg2_DR6, wherein the six residues of AnPixJg2 were entirely replaced with those of AnPixJg4, showed a 300-fold faster dark reversion than that of the wild type. We constructed chimeric proteins by fusing the GAF domains with adenylate cyclase catalytic regions, such as AnPixJg2-AC, AnPixJg4-AC and AnPixJg2_DR6-AC. We detected successful enzymatic activation under red light for both AnPixJg2-AC and AnPixJg2_DR6-AC, and repression under green light for AnPixJg2-AC and under dark incubation for AnPixJg2_DR6-AC. These results provide platforms to develop cAMP synthetic optogenetic tools.
Optogenetic control with a photocleavable protein, PhoCl.
To expand the range of experiments that are accessible with optogenetics, we developed a photocleavable protein (PhoCl) that spontaneously dissociates into two fragments after violet-light-induced cleavage of a specific bond in the protein backbone. We demonstrated that PhoCl can be used to engineer light-activatable Cre recombinase, Gal4 transcription factor, and a viral protease that in turn was used to activate opening of the large-pore ion channel Pannexin-1.
Repurposing Synechocystis PCC6803 UirS-UirR as a UV-Violet/Green Photoreversible Transcriptional Regulatory Tool in E. coli.
We have previously engineered green/red and red/far red photoreversible E. coli phytochrome and cyanobacteriochrome (CBCR) two-component systems (TCSs) and utilized them to program tailor-made gene expression signals for gene circuit characterization. Here, we transport the UV-violet/green photoreversible CBCR TCS UirS-UirR from Synechocystis PCC6803 to E. coli. We demonstrate that the promoter of the small RNA csiR1, previously shown to be activated by inorganic carbon stress, is a UirS-UirR output. Additionally, in contrast to a recently proposed sequestration model, we show that the sensor histidine kinase UirS phosphorylates the response regulator UirR to activate PcsiR1 transcription in response to UV-violet light. Finally, we measure changes in UirS-UirR output minutes after a change in light input and exploit these rapid dynamics to program a challenging gene expression signal with high predictability. UirS-UirR is the first engineered transcriptional regulatory tool activated exclusively by UV-violet light, and the most blue shifted photoreversible transcriptional regulatory tool.
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 15Z and 15E 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 15Z photostate is red-absorbing like that of phytochrome but the 15E photoproduct is instead green-absorbing. Using recombinant expression of individual CBCR domains in Escherichia coli, we fully survey the red/green subfamily from the cyanobacterium Nostoc punctiforme. 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 15E photoproduct for days, while others reverted to the 15Z 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.
Phycoviolobilin formation and spectral tuning in the DXCF cyanobacteriochrome subfamily.
Phytochromes are red/far-red photosensory proteins that regulate adaptive responses to light via photoswitching of cysteine-linked linear tetrapyrrole (bilin) chromophores. The related cyanobacteriochromes (CBCRs) extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. CBCRs and phytochromes share a conserved Cys residue required for bilin attachment. In one CBCR subfamily, often associated with a blue/green photocycle, a second Cys lies within a conserved Asp-Xaa-Cys-Phe (DXCF) motif and is essential for the blue/green photocycle. Such DXCF CBCRs use isomerization of the phycocyanobilin (PCB) chromophore into the related phycoviolobilin (PVB) to shorten the conjugated system for sensing green light. We here use recombinant expression of individual CBCR domains in Escherichia coli to survey the DXCF subfamily from the cyanobacterium Nostoc punctiforme. We describe ten new photoreceptors with well-resolved photocycles and three additional photoproteins with overlapping dark-adapted and photoproduct states. We show that the ability of this subfamily to form PVB or retain PCB provides a powerful mechanism for tuning the photoproduct absorbance, with blue-absorbing dark states leading to a broad range of photoproducts absorbing teal, green, yellow, or orange light. Moreover, we use a novel green/teal CBCR that lacks the blue-absorbing dark state to demonstrate that PVB formation requires the DXCF Cys residue. Our results demonstrate that this subfamily exhibits much more spectral diversity than had been previously appreciated.