Photoreception and signaling in bacterial phytochrome revealed by single-particle cryo-EM.

Phytochromes are red-light photoreceptors discovered in plants with homologs in bacteria and fungi that regulate a variety of physiological responses. They display a reversible photocycle between two distinct states: a red-light-absorbing Pr state and a far-red light-absorbing Pfr state. The photoconversion regulates the activity of an enzymatic domain, usually a histidine kinase (HK). The molecular mechanism that explains how light controls the HK activity is not understood because structures of unmodified bacterial phytochromes with HK activity are missing. Here, we report three cryo-electron microscopy structures of a wild-type bacterial phytochrome with HK activity determined as Pr and Pfr homodimers and as a Pr/Pfr heterodimer with individual subunits in distinct states. We propose that the Pr/Pfr heterodimer is a physiologically relevant signal transduction intermediate. Our results offer insight into the molecular mechanism that controls the enzymatic activity of the HK as part of a bacterial two-component system that perceives and transduces light signals.

Signaling by a bacterial phytochrome histidine kinase involves a conformational cascade reorganizing the dimeric photoreceptor.

Phytochromes (Phys) are a divergent cohort of bili-proteins that detect light through reversible interconversion between dark-adapted Pr and photoactivated Pfr states. While our understandings of downstream events are emerging, it remains unclear how Phys translate light into an interpretable conformational signal. Here, we present models of both states for a dimeric Phy with histidine kinase (HK) activity from the proteobacterium Pseudomonas syringae, which were built from high-resolution cryo-EM maps (2.8-3.4-Å) of the photosensory module (PSM) and its following signaling (S) helix together with lower resolution maps for the downstream output region augmented by RoseTTAFold and AlphaFold structural predictions. The head-to-head models reveal the PSM and its photointerconversion mechanism with strong clarity, while the HK region is interpretable but relatively mobile. Pr/Pfr comparisons show that bilin phototransformation alters PSM architecture culminating in a scissoring motion of the paired S-helices linking the PSMs to the HK bidomains that ends in reorientation of the paired catalytic ATPase modules relative to the phosphoacceptor histidines. This action apparently primes autophosphorylation enroute to phosphotransfer to the cognate DNA-binding response regulator AlgB which drives quorum-sensing behavior through transient association with the photoreceptor. Collectively, these models illustrate how light absorption conformationally translates into accelerated signaling by Phy-type kinases.

Red/green cyanobacteriochromes acquire isomerization from phycocyanobilin to phycoviolobilin.

Cyanobacteriochromes (CBCRs) are unique cyanobacteria-specific photoreceptors that share a distant relation with phytochromes. Most CBCRs contain conserved cysteine residues known as canonical Cys, while some CBCRs have additional cysteine residues called second Cys within the DXCF motif, leading to their classification as DXCF CBCRs. They typically undergo a process where they incorporate phycocyanobilin (PCB) and subsequently isomerize it to phycoviolobilin (PVB). Conversely, CBCRs with conserved Trp residues and without the second Cys are called extended red/green (XRG) CBCRs. Typical XRG CBCRs bind PCB without undergoing PCB-to-PVB isomerization, displaying red/green reversible photoconversion, and there are also atypical CBCRs that exhibit diverse photoconversions. We discovered novel XRG CBCRs with Cys residue instead of the conserved Trp residue. These novel XRG CBCRs exhibited the ability to isomerize PCB to PVB, displaying green/teal reversible photoconversion. Through sequence- and structure-based comparisons coupled with mutagenesis experiments, we identified three amino acid residues, including the Cys residue, crucial for facilitating PCB-to-PVB isomerization. This research expands our understanding of the diversity of XRG CBCRs, highlighting the remarkable molecular plasticity of CBCRs.

Light reception of Phycomyces revisited: several white collar proteins confer blue- and red-light sensitivity and control dynamic range and adaptation.

The giant-fruiting body, sporangiophore, of the fungus Phycomyces blakesleeanus grows toward near-UV/blue-light (phototropism). The blue-light photoreceptor, MadA, should contain FAD bound to the LOV domain, and forms a complex with MadB. Both proteins are homologs of white collar proteins WC-1 and WC-2 from the fungus Neurospora crassa and should be localized in nuclei, where they function as a light-sensitive transcription factor complex. The photoreceptor properties of two further Wc proteins, WcoA and WcoB, remain unclear because of lack of mutants. We propose that WcoA and/or WcoB play essential roles in photoreception by enlarging the dynamic range that help explain complex stimulus-response relationships. Even though red light does not elicit photo-movement or -differentiation in Phycomyces, it affects the effectiveness of blue light which indicates an underlying photochromic receptor. Protein sequence searches show that other fungal red-light receptors are absent in Phycomyces. The solution to the red-light riddle is thus sought in the ability of Wc complexes to generate after blue-light irradiation a neutral flavosemiquinone radical that absorbs red light and functions as primary photochemical signal. Phototropism requires Ras-GAP (MadC) as part of the signal transduction cascade and, we propose, to allocate photoreceptors in the plasmalemma of the growing zone, which allows for receptor dichroism, range adjustment and contrast recognition for spatial orientation. Phototropic signal chains must entail transduction networks between Wc receptors and small G-proteins and their associated Ras-GAP and Ras-GEF proteins. The interactions among these proteins should occur in trans-Golgi vesicles and the plasmalemma of the growing zone.

Photosystems and photoreceptors in cyanobacterial phototaxis and photophobotaxis.

Cyanobacteria move by gliding motility on surfaces toward the light or away from it. It is as yet unclear how the light direction is sensed on the molecular level. Diverse photoreceptor knockout mutants have a stronger response toward the light than the wild type. Either the light direction is sensed by multiple photoreceptors or by photosystems. In a study on photophobotaxis of the filamentous cyanobacterium Phormidium lacuna, broad spectral sensitivity, inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and a highly sensitive response speaks for photosystems as light direction sensors. Here, it is discussed whether the photosystem theory could hold for phototaxis of other cyanobacteria.

Teal-light absorbing cyanobacterial phytochrome superfamily provides insights into the diverse mechanisms of spectral tuning and facilitates the engineering of photoreceptors for optogenetic tools.

Cyanobacteriochromes (CBCRs) are distinctive tetrapyrrole (bilin)-binding photoreceptors exclusively found in cyanobacteria. Unlike canonical phytochromes, CBCRs require only a GAF (cGMP-phosphodiesterase/adenylate cyclase/FhlA) domain for autolyase activity to form a bilin adduct via a Cys residue and cis-trans photoisomerization. Apart from the canonical Cys, which attaches covalently to C31 in the A-ring of the bilin, some GAF domains of CBCRs contain a second-Cys in the Asp-Xaa-Cys-Phe (DXCF) motif, responsible for isomerization of phycocyanobilin (PCB) to phycoviolobilin (PVB) and/or for the formation of a reversible 2nd thioether linkage to the C10. Unlike green/teal-absorbing GAF proteins lacking ligation activity, the second-Cys in another teal-absorbing lineage (DXCF blue/teal group) exhibits both isomerization and ligation activity due to the presence of the Tyr instead of His next to the canonical Cys. Herein, we discovered an atypical CBCR GAF protein, Tpl7205g1, belonging to the DXCF blue/teal group, but having His instead of Tyr next to the first-Cys. Consistent with its subfamily, the second-Cys of Tpl7205g1 did not form a thioether linkage at C10 of PCB, showing only isomerization activity. Instead of forming 2nd thioether linkage, this novel GAF protein exhibits a pH-dependent photocycle between protonated 15Z and deprotonated 15E. Site-directed mutagenesis to the GAF scaffolds revealed its combined characteristics, including properties of teal-DXCF CBCRs and red/green-absorbing CBCRs (XRG CBCRs), suggesting itself as the evolutionary bridge between the two CBCR groups. Our study thus sheds light on the expanded spectral tuning characteristics of teal-light absorbing CBCRs and enhances feasibility of engineering these photoreceptors.

Ultrafast Primary Dynamics and Isomerization Mechanism of a Far-Red Sensing Cyanobacteriochrome.

Far-red cyanobacteriochromes (CBCRs) are bilin-based photosensory proteins that promise to be novel optical agents in optogenetics and deep tissue imaging. Recent structural studies of a far-red CBCR 2551g3 have revealed a unique all-Z,syn chromophore conformation in the far-red-absorbing Pfr state. Understanding the photoswitching mechanism through bilin photoisomerization is important for developing novel biomedical applications. Here, we employ femtosecond spectroscopy and site-directed mutagenesis to systematically characterize the dynamics of wild-type 2551g3 and four critical mutants in the 15Z Pfr state. We captured local relaxations in several picoseconds and isomerization dynamics in hundreds of picoseconds. Most mutants exhibited faster local relaxation, while their twisting dynamics and photoproducts depend on specific protein-chromophore interactions around the D-ring and C-ring. These results collectively reveal a unique dynamic pattern of excited-state evolution arising from a relatively rigid protein environment, thereby elucidating the molecular mechanism of Pfr-state photoisomerization in far-red CBCRs.

KINNTREX: a neural network to unveil protein mechanisms from time-resolved X-ray crystallography.

Here, a machine-learning method based on a kinetically informed neural network (NN) is introduced. The proposed method is designed to analyze a time series of difference electron-density maps from a time-resolved X-ray crystallographic experiment. The method is named KINNTREX (kinetics-informed NN for time-resolved X-ray crystallography). To validate KINNTREX, multiple realistic scenarios were simulated with increasing levels of complexity. For the simulations, time-resolved X-ray data were generated that mimic data collected from the photocycle of the photoactive yellow protein. KINNTREX only requires the number of intermediates and approximate relaxation times (both obtained from a singular valued decomposition) and does not require an assumption of a candidate mechanism. It successfully predicts a consistent chemical kinetic mechanism, together with difference electron-density maps of the intermediates that appear during the reaction. These features make KINNTREX attractive for tackling a wide range of biomolecular questions. In addition, the versatility of KINNTREX can inspire more NN-based applications to time-resolved data from biological macromolecules obtained by other methods.

Photoreversible Aggregation of the Biliprotein Containing the First and Second GAF Domains of a Cyanobacteriochrome All2699 in Nostoc sp. PCC7120.

As plant photoreceptors, phytochromes are capable of detecting red light and far-red light, thereby governing plant growth. All2699 is a photoreceptor found in Nostoc sp. PCC7120 that specifically responds to red light and far-red light. All2699g1g2 is a truncated protein carrying the first and second GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains of All2699. In this study, we found that, upon exposure to red light, the protein underwent aggregation, resulting in the formation of protein aggregates. Conversely, under far-red light irradiation, these protein aggregates dissociated. We delved into the factors that impact the aggregation of All2699g1g2, focusing on the protein structure. Our findings showed that the GAF2 domain contains a low-complexity (LC) loop region, which plays a crucial role in mediating protein aggregation. Specifically, phenylalanine at position 239 within the LC loop region was identified as a key site for the aggregation process. Furthermore, our research revealed that various factors, including irradiation time, temperature, concentration, NaCl concentration, and pH value, can impact the aggregation of All2699g1g2. The aggregation led to variations in Pfr concentration depending on temperature, NaCl concentration, and pH value. In contrast, ΔLC did not aggregate and therefore lacked responses to these factors. Consequently, the LC loop region of All2699g1g2 extended and enhanced sensory properties.

The LOV-domain blue-light receptor LreA of the fungus Alternaria alternata binds predominantly FAD as chromophore and acts as a light and temperature sensor.

Light and temperature sensing are important features of many organisms. Light may provide energy but may also be used by non-photosynthetic organisms for orientation in the environment. Recent evidence suggests that plant and fungal phytochrome and plant phototropin serve dual functions as light and temperature sensors. Here we characterized the fungal LOV-domain blue-light receptor LreA of Alternaria alternata and show that it predominantly contains FAD as chromophore. Blue-light illumination induced ROS production followed by protein agglomeration in vitro. In vivo ROS may control LreA activity. LreA acts as a blue-light photoreceptor but also triggers temperature-shift-induced gene expression. Both responses required the conserved amino acid cysteine 421. We therefore propose that temperature mimics the photoresponse, which could be the ancient function of the chromoprotein. Temperature-dependent gene expression control with LreA was distinct from the response with phytochrome suggesting fine-tuned, photoreceptor-specific gene regulation.

Darkness inhibits autokinase activity of bacterial bathy phytochromes.

Bathy phytochromes are a subclass of bacterial biliprotein photoreceptors that carry a biliverdin IXα chromophore. In contrast to prototypical phytochromes that adopt a red-light-absorbing Pr ground state, the far-red light-absorbing Pfr-form is the thermally stable ground state of bathy phytochromes. Although the photobiology of bacterial phytochromes has been extensively studied since their discovery in the late 1990s, our understanding of the signal transduction process to the connected transmitter domains, which are often histidine kinases, remains insufficient. Initiated by the analysis of the bathy phytochrome PaBphP from Pseudomonas aeruginosa, we performed a systematic analysis of five different bathy phytochromes with the aim to derive a general statement on the correlation of photostate and autokinase output. While all proteins adopt different Pr/Pfr-fractions in response to red, blue, and far-red light, only darkness leads to a pure or highly enriched Pfr-form, directly correlated with the lowest level of autokinase activity. Using this information, we developed a method to quantitatively correlate the autokinase activity of phytochrome samples with well-defined stationary Pr/Pfr-fractions. We demonstrate that the off-state of the phytochromes is the Pfr-form and that different Pr/Pfr-fractions enable the organisms to fine-tune their kinase output in response to a certain light environment. Furthermore, the output response is regulated by the rate of dark reversion, which differs significantly from 5 s to 50 min half-life. Overall, our study indicates that bathy phytochromes function as sensors of light and darkness, rather than red and far-red light, as originally postulated.

Conserved Signal Transduction Mechanisms and Dark Recovery Kinetic Tuning in the Pseudomonadaceae Short Light, Oxygen, Voltage (LOV) Protein Family.

Light-Oxygen-Voltage (LOV) flavoproteins transduce a light signal into variable signaling outputs via a structural rearrangement in the sensory core domain, which is then relayed to fused effector domains via α-helical linker elements. Short LOV proteins from Pseudomonadaceae consist of a LOV sensory core and N- and C-terminal α-helices of variable length, providing a simple model system to study the molecular mechanism of allosteric activation. Here we report the crystal structures of two LOV proteins from Pseudomonas fluorescens - SBW25-LOV in the fully light-adapted state and Pf5-LOV in the dark-state. In a comparative analysis of the Pseudomonadaceae short LOVs, the structures demonstrate light-induced rotation of the core domains and splaying of the proximal A'α and Jα helices in the N and C-termini, highlighting evidence for a conserved signal transduction mechanism. Another distinguishing feature of the Pseudomonadaceae short LOV protein family is their highly variable dark recovery, ranging from seconds to days. Understanding this variability is crucial for tuning the signaling behavior of LOV-based optogenetic tools. At 37 °C, SBW25-LOV and Pf5-LOV exhibit adduct state lifetimes of 1470 min and 3.6 min, respectively. To investigate this remarkable difference in dark recovery rates, we targeted three residues lining the solvent channel entrance to the chromophore pocket where we introduced mutations by exchanging the non-conserved amino acids from SBW25-LOV into Pf5-LOV and vice versa. Dark recovery kinetics of the resulting mutants, as well as MD simulations and solvent cavity calculations on the crystal structures suggest a correlation between solvent accessibility and adduct lifetime.

Introduction of reversible cysteine ligation ability to the biliverdin-binding cyanobacteriochrome photoreceptor.

Cyanobacteriochrome (CBCR) photoreceptors are distantly related to the canonical red/far-red reversible phytochrome photoreceptors. In the case of the CBCRs, only the GAF domain is required for chromophore incorporation and photoconversion. The GAF domains of CBCR are highly diversified into many lineages to sense various colors of light. These CBCR GAF domains are divided into two types: those possessing only the canonical Cys residue and those with both canonical and second Cys residues. The canonical Cys residue stably ligates to the chromophore in both cases. The second Cys residue mostly shows reversible adduct formation with the chromophore during photoconversion for spectral tuning. In this study, we focused on the CBCR GAF domain AnPixJg2_BV4, which possesses only the canonical Cys residue. AnPixJg2_BV4 covalently ligates to the biliverdin (BV) chromophore and shows far-red/orange reversible photoconversion. Because BV is a mammalian intrinsic chromophore, BV-binding molecules are advantageous for in vivo optogenetic and bioimaging tool development. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis and serendipitously obtained a unique variant molecule that showed far-red/blue reversible photoconversion, in which the Cys residue was introduced near the chromophore. This introduced Cys residue functioned as the second Cys residue that reversibly ligated with the chromophore. Because the position of the introduced Cys residue is distinct from the known second Cys residues, the variant molecule obtained in this study would expand our knowledge about the spectral tuning mechanism of CBCRs and contribute to tool development.

Photoactive Yellow Protein Represents a Distinct, Evolutionarily Novel Family of PAS Domains.

Photoactive yellow protein (PYP) is a model photoreceptor. It binds a p-coumaric acid as a chromophore, thus enabling blue light sensing. The first discovered single-domain PYP from Halorhodospira halophila has been studied thoroughly in terms of its structural dynamics and photochemical properties. However, the evolutionary origins and biological role of PYP homologs are not well understood. Here, we show that PYP is an evolutionarily novel domain family of the ubiquitous PAS (Per-Arnt-Sim) superfamily. It likely originated from the phylum Myxococcota and was then horizontally transferred to representatives of a few other bacterial phyla. We show that PYP is associated with signal transduction either by domain fusion or by genome context. Key cellular functions modulated by PYP-initiated signal transduction pathways likely involve gene expression, motility, and biofilm formation. We identified three clades of the PYP family, one of which is poorly understood and potentially has novel functional properties. The Tyr42, Glu46, and Cys69 residues that are involved in p-coumaric acid binding in the model PYP from H. halophila are well conserved in the PYP family. However, we also identified cases where substitutions in these residues might have led to neofunctionalization, such as the proposed transition from light to redox sensing. Overall, this study provides definition, a newly built hidden Markov model, and the current genomic landscape of the PYP family and sets the stage for the future exploration of its signaling mechanisms and functional diversity. IMPORTANCE Photoactive yellow protein is a model bacterial photoreceptor. For many years, it was considered a prototypical model of the ubiquitous PAS domain superfamily. Here, we show that, in fact, the PYP family is evolutionarily novel, restricted to a few bacterial phyla and distinct from other PAS domains. We also reveal the diversity of PYP-containing signal transduction proteins and their potential mechanisms.

Light-induced production of isobutanol and 3-methyl-1-butanol by metabolically engineered cyanobacteria.

BACKGROUND: Cyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully produced in engineered cyanobacteria. Chemical inducer-dependent promoters are used to induce the expression of target biosynthetic pathway genes. A chemical inducer is not ideal for large-scale reactions owing to its high cost; therefore, it is important to develop scaling-up methods to avoid their use. In this study, we designed a green light-inducible alcohol production system using the CcaS/CcaR green light gene expression system in the cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803). RESULTS: To establish the green light-inducible production of isobutanol and 3-methyl-1-butanol (3MB) in PCC 6803, keto-acid decarboxylase (kdc) and alcohol dehydrogenase (adh) were expressed under the control of the CcaS/CcaR system. Increases in the transcription level were induced by irradiation with red and green light without severe effects on host cell growth. We found that the production of isobutanol and 3MB from carbon dioxide (CO2) was induced under red and green light illumination and was substantially repressed under red light illumination alone. Finally, production titers of isobutanol and 3MB reached 238 mg L-1 and 75 mg L-1, respectively, in 5 days under red and green light illumination, and these values are comparable to those reported in previous studies using chemical inducers. CONCLUSION: A green light-induced alcohol production system was successfully integrated into cyanobacteria to produce value-added chemicals without using expensive chemical inducers. The green light-regulated production of isobutanol and 3MB from CO2 is eco-friendly and cost-effective. This study demonstrates that light regulation is a potential tool for producing chemicals and increases the feasibility of cyanobacterial bioprocesses.

Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane.

Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins.

Few-fs resolution of a photoactive protein traversing a conical intersection.

The structural dynamics of a molecule are determined by the underlying potential energy landscape. Conical intersections are funnels connecting otherwise separate potential energy surfaces. Posited almost a century ago1, conical intersections remain the subject of intense scientific interest2-5. In biology, they have a pivotal role in vision, photosynthesis and DNA stability6. Accurate theoretical methods for examining conical intersections are at present limited to small molecules. Experimental investigations are challenged by the required time resolution and sensitivity. Current structure-dynamical understanding of conical intersections is thus limited to simple molecules with around ten atoms, on timescales of about 100 fs or longer7. Spectroscopy can achieve better time resolutions8, but provides indirect structural information. Here we present few-femtosecond, atomic-resolution videos of photoactive yellow protein, a 2,000-atom protein, passing through a conical intersection. These videos, extracted from experimental data by machine learning, reveal the dynamical trajectories of de-excitation via a conical intersection, yield the key parameters of the conical intersection controlling the de-excitation process and elucidate the topography of the electronic potential energy surfaces involved.

Ultrafast Photoconversion Dynamics of the Knotless Phytochrome SynCph2.

The family of phytochrome photoreceptors contains proteins with different domain architectures and spectral properties. Knotless phytochromes are one of the three main subgroups classified by their distinct lack of the PAS domain in their photosensory core module, which is in contrast to the canonical PAS-GAF-PHY array. Despite intensive research on the ultrafast photodynamics of phytochromes, little is known about the primary kinetics in knotless phytochromes. Here, we present the ultrafast Pr ⇆ Pfr photodynamics of SynCph2, the best-known knotless phytochrome. Our results show that the excited state lifetime of Pr* (~200 ps) is similar to bacteriophytochromes, but much longer than in most canonical phytochromes. We assign the slow Pr* kinetics to relaxation processes of the chromophore-binding pocket that controls the bilin chromophore's isomerization step. The Pfr photoconversion dynamics starts with a faster excited state relaxation than in canonical phytochromes, but, despite the differences in the respective domain architectures, proceeds via similar ground state intermediate steps up to Meta-F. Based on our observations, we propose that the kinetic features and overall dynamics of the ultrafast photoreaction are determined to a great extent by the geometrical context (i.e., available space and flexibility) within the binding pocket, while the general reaction steps following the photoexcitation are most likely conserved among the red/far-red phytochromes.

Structure-based design of a photoswitchable affibody scaffold.

Photo-control of affinity reagents offers a general approach for high-resolution spatiotemporal control of diverse molecular processes. In an effort to develop general design principles for a photo-controlled affinity reagent, we took a structure-based approach to the design of a photoswitchable Z-domain, among the simplest of affinity reagent scaffolds. A chimera, designated Z-PYP, of photoactive yellow protein (PYP) and the Z-domain, was designed based on the concept of mutually exclusive folding. NMR analysis indicated that, in the dark, the PYP domain of the chimera was folded, and the Z-domain was unfolded. Blue light caused loss of structure in PYP and a two- to sixfold change in the apparent affinity of Z-PYP for its target as determined using size exclusion chromatography, UV-Vis based assays, and enyzme-linked immunosorbent assay (ELISA). A thermodynamic model indicated that mutations to decrease Z-domain folding energy would alter target affinity without loss of switching. This prediction was confirmed experimentally with a double alanine mutant in helix 3 of the Z-domain of the chimera (Z-PYP-AA) showing >30-fold lower dark-state binding and no loss in switching. The effect of decreased dark-state binding affinity was tested in a two-hybrid transcriptional control format and enabled pronounced light/dark differences in yeast growth in vivo. Finally, the design was transferable to the αZ-Taq affibody enabling tunable light-dependent binding both in vitro and in vivo to the Z-Taq target. This system thus provides a framework for the focused development of light switchable affibodies for a range of targets.

A guide to the optogenetic regulation of endogenous molecules.

Genetically encoded tools for the regulation of endogenous molecules (RNA, DNA elements and protein) are needed to study and control biological processes with minimal interference caused by protein overexpression and overactivation of signaling pathways. Here we focus on light-controlled optogenetic tools (OTs) that allow spatiotemporally precise regulation of gene expression and protein function. To control endogenous molecules, OTs combine light-sensing modules from natural photoreceptors with specific protein or nucleic acid binders. We discuss OT designs and group OTs according to the principles of their regulation. We outline characteristics of OT performance, discuss considerations for their use in vivo and review available OTs and their applications in cells and in vivo. Finally, we provide a brief outlook on the development of OTs.