Curated Optogenetic Publication Database

Abstract: Blue light sensor using flavin (BLUF) proteins consist of flavin-binding BLUF domains and functional domains. Upon blue light excitation, the hydrogen bond network around the flavin chromophore changes, and the absorption spectrum in the visible region exhibits a red shift. Ultimately, the light information received in the BLUF domain is transmitted to the functional region. It has been believed that this red shift is complete within nanoseconds. In this study, slow reaction kinetics were discovered in milliseconds (τ1- and τ2-phase) for all the BLUF proteins examined (AppA, OaPAC, BlrP1, YcgF, PapB, SyPixD, and TePixD). Despite extensive reports on BLUF, this is the first clear observation of the BLUF protein absorption change with the duration in the millisecond time region. From the measurements of some domain-deleted mutants of OaPAC and two chimeric mutants of PixD proteins, it was found that the slower dynamics (τ2-phase) are strongly affected by the size and nature of the C-terminal region adjacent to the BLUF domain. Hence, this millisecond reaction is a significant indicator of conformational changes in the C-terminal region, which is essential for the biological functions. On the other hand, the τ1-phase commonly exists in all BLUF proteins, including any mutants. The origin of the slow dynamics was studied using site-specific mutants. These results clearly show the importance of Trp in the BLUF domain. Based on this, a reaction scheme for the BLUF reaction is proposed.

Abstract: The SpyCatcher/SpyTag protein conjugation system has recently exploded in popularity due to its fast kinetics and high yield under biologically favorable conditions in both in vitro and intracellular settings. The utility of this system could be expanded by introducing the ability to spatially and temporally control the conjugation event. Taking inspiration from photoreceptor proteins in nature, we designed a method to integrate light dependency into the protein conjugation reaction. The light-oxygen-voltage domain 2 of Avena sativa (AsLOV2) undergoes a dramatic conformational change in its c-terminal Jα-helix in response to blue light. By inserting SpyTag into the different locations of the Jα-helix, we created a blue light inducible SpyTag system (BLISS). In this design, the SpyTag is blocked from reacting with the SpyCatcher in the dark, but upon irradiation with blue light, the Jα-helix of the AsLOV2 undocks to expose the SpyTag. We tested several insertion sites and characterized the kinetics. We found three variants with dynamic ranges over 15, which were active within different concentration ranges. These could be tuned using SpyCatcher variants with different reaction kinetics. Further, the reaction could be instantaneously quenched by removing light. We demonstrated the spatial aspect of this light control mechanism through photopatterning of two fluorescent proteins. This system offers opportunities for many other biofabrication and optogenetics applications.

Abstract: Compartmentation of proteins into biomolecular condensates or membraneless organelles formed by phase separation is an emerging principle for the regulation of cellular processes. Creating synthetic condensates that accommodate specific intracellular proteins on demand would have various applications in chemical biology, cell engineering, and synthetic biology. Here, we report the construction of synthetic protein condensates capable of recruiting and/or releasing proteins of interest in living mammalian cells in response to a small molecule or light. By a modular combination of a tandem fusion of two oligomeric proteins, which forms phase-separated synthetic protein condensates in cells, with a chemically induced dimerization tool, we first created a chemogenetic protein condensate system that can rapidly recruit target proteins from the cytoplasm to the condensates by addition of a small-molecule dimerizer. We next coupled the protein-recruiting condensate system with an engineered proximity-dependent protease, which gave a second protein condensate system wherein target proteins previously expressed inside the condensates are released into the cytoplasm by small-molecule-triggered protease recruitment. Furthermore, an optogenetic condensate system that allows reversible release and sequestration of protein activity in a repeatable manner using light was constructed successfully. These condensate systems were applicable to control protein activity and cellular processes such as membrane ruffling and ERK signaling in a time scale of minutes. This proof-of-principle work provides a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells and represents a step toward tailor-made engineering of synthetic protein condensate-based soft materials with various functionalities for biological and biomedical applications.

Abstract: 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.

Abstract: Light-oxygen-voltage (LOV) domains sense blue light through the photochemical formation of a cysteinyl-flavin covalent adduct. Concurrent protonation at the flavin N5 position alters the hydrogen bonding interactions of an invariant Gln residue that has been proposed to flip its amide side chain as a critical step in the propagation of conformational change. Traditional molecular dynamics (MD) and replica-exchange MD (REMD) simulations of the well-characterized LOV protein Vivid (VVD) demonstrate that the Gln182 amide indeed reorients by ∼180° in response to either adduct formation or reduction of the isoalloxazine ring to the neutral semiquinone, both of which involve N5 protonation. Free energy simulations reveal that the relative free energies of the flipped Gln conformation and the flipping barrier are significantly lower in the light-adapted state. The Gln182 flip stabilizes an important hinge-bβ region between the PAS β-sheet and the N-terminal cap helix that in turn destabilizes an N-terminal latch region against the PAS core. Release of the latch, observed both experimentally and in the simulations, is known to mediate light-induced VVD dimerization. This computational study of a LOV protein, unprecedented in its agreement with experiment, provides an atomistic view of long-range allosteric coupling in a photoreceptor.

Abstract: Living systems are fundamentally dependent on the ability of proteins to respond to external stimuli. The mechanism, the underlying structural dynamics, and the time scales for regulation of this response are central questions in biochemistry. Here we probe the structural dynamics of the BLUF domain found in several photoactive flavoproteins, which is responsible for light activated functions as diverse as phototaxis and gene regulation. Measurements have been made over 10 decades of time (from 100 fs to 1 ms) using transient vibrational spectroscopy. Chromophore (flavin ring) localized dynamics occur on the pico- to nanosecond time scale, while subsequent protein structural reorganization is observed over microseconds. Multiple time scales are observed for the dynamics associated with different vibrations of the protein, suggesting an underlying hierarchical relaxation pathway. Structural evolution in residues directly H-bonded to the chromophore takes place more slowly than changes in more remote residues. However, a point mutation which suppresses biological function is shown to 'short circuit' this structural relaxation pathway, suppressing the changes which occur further away from the chromophore while accelerating dynamics close to it.

Abstract: Advanced gene regulatory systems are necessary for scientific research, synthetic biology, and gene-based medicine. An ideal system would allow facile spatiotemporal manipulation of gene expression within a cell population that is tunable, reversible, repeatable, and can be targeted to diverse DNA sequences. To meet these criteria, a gene regulation system was engineered that combines light-sensitive proteins and programmable zinc finger transcription factors. This system, light-inducible transcription using engineered zinc finger proteins (LITEZ), uses two light-inducible dimerizing proteins from Arabidopsis thaliana, GIGANTEA and the LOV domain of FKF1, to control synthetic zinc finger transcription factor activity in human cells. Activation of gene expression in human cells engineered with LITEZ was reversible and repeatable by modulating the duration of illumination. The level of gene expression could also be controlled by modulating light intensity. Finally, gene expression could be activated in a spatially defined pattern by illuminating the human cell culture through a photomask of arbitrary geometry. LITEZ enables new approaches for precisely regulating gene expression in biotechnology and medicine, as well as studying gene function, cell-cell interactions, and tissue morphogenesis.

Abstract: PixD (Slr1694) is a blue light receptor that contains a BLUF (blue light sensors using a flavin chromophore) domain. A protein-protein interaction between PixD and a response regulator PixE (Slr1693) is essential to achieve light signal transduction for phototaxis of the species. Although the initial photochemical reaction of PixD, the red shift of the flavin absorption spectrum, has been investigated, the subsequent reaction dynamics remain largely unresolved. Only the disassembly of the PixD(10)-PixE(5) dark complex has been characterized by static size exclusion chromatography. In this report, interprotein reaction dynamics were examined using time-resolved transient grating spectroscopy. The dissociation process was clearly observed as the light-induced diffusion coefficient change in the time domain, and the kinetics was determined. More strikingly, disassembly was found to take place only after photoactivation of two PixD subunits in the complex. This result suggests that the biological response of PixD does not follow a linear correlation with the light intensity but appears to be light-intensity-dependent.

Abstract: The signaling state of the photoactive yellow protein (PYP) photoreceptor is transiently developed via isomerization of its blue-light-absorbing chromophore. The associated structural rearrangements have large amplitude but, due to its transient nature and chemical exchange reactions that complicate NMR detection, its accurate three-dimensional structure in solution has been elusive. Here we report on direct structural observation of the transient signaling state by combining double electron electron resonance spectroscopy (DEER), NMR, and time-resolved pump-probe X-ray solution scattering (TR-SAXS/WAXS). Measurement of distance distributions for doubly spin-labeled photoreceptor constructs using DEER spectroscopy suggests that the signaling state is well ordered and shows that interspin-label distances change reversibly up to 19 Å upon illumination. The SAXS/WAXS difference signal for the signaling state relative to the ground state indicates the transient formation of an ordered and rearranged conformation, which has an increased radius of gyration, an increased maximum dimension, and a reduced excluded volume. Dynamical annealing calculations using the DEER derived long-range distance restraints in combination with short-range distance information from (1)H-(15)N HSQC perturbation spectroscopy give strong indication for a rearrangement that places part of the N-terminal domain in contact with the exposed chromophore binding cleft while the terminal residues extend away from the core. Time-resolved global structural information from pump-probe TR-SAXS/WAXS data supports this conformation and allows subsequent structural refinement that includes the combined energy terms from DEER, NMR, and SAXS/WAXS together. The resulting ensemble simultaneously satisfies all restraints, and the inclusion of TR-SAXS/WAXS effectively reduces the uncertainty arising from the possible spin-label orientations. The observations are essentially compatible with reduced folding of the I(2)' state (also referred to as the 'pB' state) that is widely reported, but indicates it to be relatively ordered and rearranged. Furthermore, there is direct evidence for the repositioning of the N-terminal region in the I(2)' state, which is structurally modeled by dynamical annealing and refinement calculations.