Analysis of Slow-Cycling Variants of the Light-Inducible Nuclear Protein Export System LEXY in Mammalian Cells.

The optogenetic tool LEXY consists of the second light oxygen voltage (LOV) domain of Avena sativa phototropin 1 mutated to contain a nuclear export signal. It allows exporting from the nucleus with blue light proteins of interest (POIs) genetically fused to it. Mutations slowing the dark recovery rate of the LOV domain within LEXY were recently shown to allow for better depletion of some POIs from the nucleus in Drosophila embryos and for the usage of low light illumination regimes. We investigated these variants in mammalian cells and found they increase the cytoplasmic localization of the proteins we tested after illumination, but also during the dark phases, which corresponds to higher leakiness of the system. These data suggest that, when aiming to sequester into the nucleus a protein with a cytoplasmic function, the original LEXY is preferable. The iLEXY variants are, instead, advantageous when wanting to deplete the nucleus of the POI as much as possible.

Out of the blue: Phototropins of the leaf vascular bundle sheath mediate the regulation of leaf hydraulic conductance by blue light.

The Arabidopsis (Arabidopsis thaliana) leaf veins bundle-sheath cells (BSCs)-a selective barrier to water and solutes entering the mesophyll-increase the leaf radial hydraulic conductance (Kleaf) by acidifying the xylem sap by their plasma membrane H+-ATPase,  AHA2. Based on this and on the BSCs' expression of phototropins PHOT1 and PHOT2, and the known blue light (BL)-induced Kleaf increase, we hypothesized that, resembling the guard cells, BL perception by the BSCs' phots activates its H+-ATPase, which, consequently, upregulates Kleaf. Indeed, under BL, the Kleaf of the knockout mutant lines phot1-5, phot2-1, phot1-5 phot2-1, and aha2-4 was lower than that of the wild-type (WT). BSC-only-directed complementation of phot1-5 or aha2-4 by PHOT1 or AHA2, respectively, restored the BL-induced Kleaf increase. BSC-specific silencing of PHOT1 or PHOT2 prevented such Kleaf increase. A xylem-fed kinase inhibitor (tyrphostin 9) replicated this also in WT plants. White light-ineffective in the phot1-5 mutant-acidified the xylem sap (relative to darkness) in WT and in the PHOT1-complemented phot1-5. These results, supported by BL increase of BSC protoplasts' water permeability and cytosolic pH and their hyperpolarization by BL, identify the BSCs as a second phot-controlled water conductance element in leaves, in series with stomatal conductance. Through both, BL regulates the leaf water balance.

Minimally disruptive optical control of protein tyrosine phosphatase 1B.

Protein tyrosine phosphatases regulate a myriad of essential subcellular signaling events, yet they remain difficult to study in their native biophysical context. Here we develop a minimally disruptive optical approach to control protein tyrosine phosphatase 1B (PTP1B)-an important regulator of receptor tyrosine kinases and a therapeutic target for the treatment of diabetes, obesity, and cancer-and we use that approach to probe the intracellular function of this enzyme. Our conservative architecture for photocontrol, which consists of a protein-based light switch fused to an allosteric regulatory element, preserves the native structure, activity, and subcellular localization of PTP1B, affords changes in activity that match those elicited by post-translational modifications inside the cell, and permits experimental analyses of the molecular basis of optical modulation. Findings indicate, most strikingly, that small changes in the activity of PTP1B can cause large shifts in the phosphorylation states of its regulatory targets.

Quantification of light-induced miniSOG superoxide production using the selective marker, 2-hydroxyethidium.

Genetically-encoded photosensitizers produce reactive oxygen species (ROS) in response to light. Transgenic expression of fusion proteins can target the photosensitizers to specific cell regions and permit the spatial and temporal control of ROS production. These ROS-generating proteins (RGPs) are widely used for cell ablation, mutagenesis and chromophore-assisted light inactivation of target proteins. However, the species produced by RGPs are unclear due to indirect measures with confounding interpretations. Recently, the RGP mini "Singlet Oxygen Generator" (miniSOG) was engineered from Arabidopsis thaliana phototropin 2. While miniSOG produces singlet oxygen (1O2), the contribution of superoxide (O2•-) to miniSOG-generated ROS remains unclear. We measured the light-dependent O2•- production of purified miniSOG using HPLC separation of dihydroethidium (DHE) oxidation products. We demonstrate that DHE is insensitive to 1O2 and establish that DHE is a suitable indicator to measure O2•- production in a system that produces both 1O2 and O2•-. We report that miniSOG produces both 1O2 and O2•-, as can its free chromophore, flavin mononucleotide. miniSOG produced O2•- at a rate of ~4.0µmol O2•-/min/µmol photosensitizer for an excitation fluence rate of 5.9mW/mm2 at 470 ± 20nm, and the rate remained consistent across fluences (light doses). Overall, the contribution of O2•- to miniSOG phenotypes should be considered.

Switching from adduct formation to electron transfer in a light-oxygen-voltage domain containing the reactive cysteine.

LOV (light-, oxygen- or voltage-sensitive) domains act as photosensory units of many prokaryotic and eukaryotic proteins. Upon blue light excitation they undergo a photocycle via the excited triplet state of their flavin chromophore yielding the flavin-cysteinyl adduct. Adduct formation is highly conserved among all LOV domains and constitutes the primary step of LOV domain signaling. But recently, it has been shown that signal propagation can also be triggered by flavin photoreduction to the neutral semiquinone offering new prospects for protein engineering. This, however, requires mutation of the photo-active Cys. Here, we report on LOV1 mutants of C. reinhardtii phototropin in which adduct formation is suppressed although the photo-active Cys is present. Introduction of a Tyr into the LOV core induces a proton coupled electron transfer towards the flavin chromophore. Flavin radical species are formed via either the excited flavin singlet or triplet state depending on the geometry of donor and acceptor. This photoreductive pathway resembles the photoreaction observed in other blue light photoreceptors, e.g. blue-light sensors using flavin adenine dinucleotide (BLUF) domains or cryptochromes. The ability to tune the photoreactivity of the flavin chromophore inside the LOV core has implications for the mechanism of adduct formation in the wild type and may be of use for protein engineering.

Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa LOV Domain.

The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatiotemporal control of cell signaling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds time scale, which then modulate the activity of output domains responsible for biological signaling. Using time-resolved vibrational spectroscopy coupled with isotope labeling, we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 fs and 1 ms after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labeled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a subpicosecond perturbation of the protein matrix occurs. In this perturbed environment, the previously characterized reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the ß-sheet and then α-helix regions of the AsLOV2 domain, which ultimately gives rise to Jα-helix unfolding, yielding the signaling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513.

Applications of genetically encoded photosensitizer miniSOG: from correlative light electron microscopy to immunophotosensitizing.

Genetically encoded photosensitizers (PSs), e.g. ROS generating proteins, correspond to a novel class of PSs that are highly desirable for biological and medical applications since they can be used in combination with a variety of genetic engineering manipulations allowing for precise spatio-temporal control of ROS production within living cells and organisms. In contrast to the commonly used chemical PSs, they can be modified using genetic engineering approaches and targeted to particular cellular compartments and cell types. Mini Singlet Oxygen Generator (miniSOG), a small flavoprotein capable of singlet oxygen production upon blue light irradiation, was initially reported as a high contrast probe for correlative light electron microscopy (CLEM) without the need of exogenous ligands, probes or destructive permeabilizing detergents. Further miniSOG was successfully applied for chromophore-assisted light inactivation (CALI) of proteins, as well as for photo-induced cell ablation in tissue cultures and in Caenorhabditis elegans. Finally, a novel approach of immunophotosensitizing has been developed, exploiting the specificity of mini-antibodies or selective scaffold proteins and photo-induced cytotoxicity of miniSOG, which is particularly promising for selective non-invasive photodynamic therapy of cancer (PDT) due to the spatial selectivity and locality of destructive action compared to other methods of oncotherapy.

[Preparation of Recombinant Human Adenoviruses Labeled with miniSOG].

We wished to study the intracellular transport of adenoviruses. We constructed a novel recombinant adenovirus in which the structural protein IX was labeled with a mini-singlet oxygen generator (miniSOG). The miniSOG gene was synthesized by overlapping extension polymerase chain reaction (PCR), cloned to the pcDNA3 vector, and expressed in 293 cells. Activation of miniSOG generated sufficient numbers of singlet oxygen molecules to catalyze polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by transmission electron microscopy (TEM). To construct miniSOG-labelled recombinant adenoviruses, the miniSOG gene was subcloned downstream of the IX gene in a pShuttle plasmid. Adenoviral plasmid pAd5-IXSOG was generated by homologous recombination of the modified shuttle plasmid (pShuttle-IXSOG) with the backbone plasmid (pAdeasy-1) in the BJ5183 strain of Eschericia coli. Adenovirus HAdV-5-IXSOG was rescued by transfection of 293 cells with the linearized pAd5-IXSOG. After propagation, virions were purified using the CsC1 ultracentrifugation method. Finally, HAdV-5-IXSOG in 2.0 mL with a particle titer of 6 x 1011 vp/mL was obtained. Morphology of HAdV-5-IXSOG was verified by TEM. Fusion of IX with the miniSOG gene was confirmed by PCR. In conclusion, miniSOG-labeled recombinant adenoviruses were constructed, which could be valuable tools for virus tracking by TEM.

Arabidopsis phot1 and phot2 phosphorylate BLUS1 kinase with different efficiencies in stomatal opening.

In Arabidopsis thaliana, phototropins (phot1 and phot2), light-activated receptor kinases, redundantly regulate various photoresponses such as phototropism, chloroplast photorelocation movement, stomatal opening, and leaf flattening. However, it is still unclear how phot1 and phot2 signals are integrated into a common target and regulate physiological responses. In the present study, we provide evidence that phot1 and phot2 phosphorylate BLUE LIGHT SIGNALING1 (BLUS1) kinase as a common substrate in stomatal opening. Biochemical analysis revealed that the recombinant phot2 protein directly phosphorylated BLUS1 in vitro in a blue light-dependent manner, as reported for phot1. BLUS1 phosphorylation was observed in both phot1 and phot2 mutants, and phot2 mutant exhibited higher phosphorylation of BLUS1 than did phot1 mutant. Transgenic plants expressing phot1-GFP (P1G) and phot2-GFP (P2G) at a similar level under the PHOT2 promoter demonstrated that P1G initiated higher phosphorylation of BLUS1 than P2G, suggesting that phot1 phosphorylates BLUS1 more efficiently. Similarly, P1G mediated a higher activation of the plasma membrane H(+)-ATPase and stomatal opening than P2G, indicating that the phosphorylation status of BLUS1 is a key determinant of physiological response. Together, these findings provide insights into the signal integration and different properties of phot1 and phot2 signaling.

Auxin and chloroplast movements.

Auxin is involved in a wide spectrum of physiological processes in plants, including responses controlled by the blue light photoreceptors phototropins: phototropic bending and stomatal movement. However, the role of auxin in phototropin-mediated chloroplast movements has never been studied. To address this question we searched for potential interactions between auxin and the chloroplast movement signaling pathway using different experimental approaches and two model plants, Arabidopsis thaliana and Nicotiana tabacum. We observed that the disturbance of auxin homeostasis by shoot decapitation caused a decrease in chloroplast movement parameters, which could be rescued by exogenous auxin application. In several cases, the impairment of polar auxin transport, by chemical inhibitors or in auxin carrier mutants, had a similar negative effect on chloroplast movements. This inhibition was not correlated with changes in auxin levels. Chloroplast relocations were also affected by the antiauxin p-chlorophenoxyisobutyric acid and mutations in genes encoding some of the elements of the SCF(TIR1)-Aux/IAA auxin receptor complex. The observed changes in chloroplast movement parameters are not prominent, which points to a modulatory role of auxin in this process. Taken together, the obtained results suggest that auxin acts indirectly to regulate chloroplast movements, presumably by regulating gene expression via the SCF(TIR1)-Aux/IAA-ARF pathway. Auxin does not seem to be involved in controlling the expression of phototropins.

A phytochrome/phototropin chimeric photoreceptor of fern functions as a blue/far-red light-dependent photoreceptor for phototropism in Arabidopsis.

In the fern Adiantum capillus-veneris, the phototropic response of the protonemal cells is induced by blue light and partially inhibited by subsequent irradiation with far-red light. This observation strongly suggests the existence of a phytochrome that mediates this blue/far-red reversible response; however, the phytochrome responsible for this response has not been identified. PHY3/NEO1, one of the three phytochrome genes identified in Adiantum, encodes a chimeric photoreceptor composed of both a phytochrome and a phototropin domain. It was demonstrated that phy3 mediates the red light-dependent phototropic response of Adiantum, and that phy3 potentially functions as a phototropin. These findings suggest that phy3 is the phytochrome that mediates the blue/far-red response in Adiantum protonemata. In the present study, we expressed Adiantum phy3 in a phot1 phot2 phototropin-deficient Arabidopsis line, and investigated the ability of phy3 to induce phototropic responses under various light conditions. Blue light irradiation clearly induced a phototropic response in the phy3-expressing transgenic seedlings, and this effect was fully inhibited by simultaneous irradiation with far-red light. In addition, experiments using amino acid-substituted phy3 indicated that FMN-cysteinyl adduct formation in the light, oxygen, voltage (LOV) domain was not necessary for the induction of blue light-dependent phototropism by phy3. We thus demonstrate that phy3 is the phytochrome that mediates the blue/far-red reversible phototropic response in Adiantum. Furthermore, our results imply that phy3 can function as a phototropin, but that it acts principally as a phytochrome that mediates both the red/far-red and blue/far-red light responses.

Plasma membrane H⁺ -ATPase regulation is required for auxin gradient formation preceding phototropic growth.

Phototropism is a growth response allowing plants to align their photosynthetic organs toward incoming light and thereby to optimize photosynthetic activity. Formation of a lateral gradient of the phytohormone auxin is a key step to trigger asymmetric growth of the shoot leading to phototropic reorientation. To identify important regulators of auxin gradient formation, we developed an auxin flux model that enabled us to test in silico the impact of different morphological and biophysical parameters on gradient formation, including the contribution of the extracellular space (cell wall) or apoplast. Our model indicates that cell size, cell distributions, and apoplast thickness are all important factors affecting gradient formation. Among all tested variables, regulation of apoplastic pH was the most important to enable the formation of a lateral auxin gradient. To test this prediction, we interfered with the activity of plasma membrane H⁺ -ATPases that are required to control apoplastic pH. Our results show that H⁺ -ATPases are indeed important for the establishment of a lateral auxin gradient and phototropism. Moreover, we show that during phototropism, H⁺ -ATPase activity is regulated by the phototropin photoreceptors, providing a mechanism by which light influences apoplastic pH.

Manipulation of endogenous kinase activity in living cells using photoswitchable inhibitory peptides.

Optogenetic control of endogenous signaling can be an important tool for probing cell behavior. Using the photoresponse of the LOV2 domain of Avena sativa phototropin 1, we developed analogues of kinase inhibitors whose activity is light dependent. Inhibitory peptides were appended to the Jα helix, where they potently inhibited kinases in the light but were sterically blocked from kinase interaction in the dark. Photoactivatable inhibitors for cyclic-AMP dependent kinase (PKA) and myosin light chain kinase (MLCK) are described, together with studies that shed light on proper positioning of the peptides in the LOV domain. These inhibitors altered endogenous signaling in living cells and produced light-dependent changes in cell morphodynamics.

Photochemistry of Arabidopsis phototropin 1 LOV1: transient tetramerization.

The photochemical reaction of the LOV1 (light-oxygen-voltage 1) domain of phototropin 1 from Arabidopsis thaliana was investigated by the time-resolved transient grating method. As with other LOV domains, an absorption spectral change associated with an adduct formation between its chromophore (flavin mononucleotide) and a cysteine residue was observed with a time constant of 1.1 µs. After this reaction, a significant diffusion coefficient (D) change (D of the reactant = 8.2 × 10(-11) m(2) s(-1), and D of the photoproduct = 6.4 × 10(-11) m(2) s(-1)) was observed with a time constant of 14 ms at a protein concentration of 270 µM. From the D value of the ground state and the peak position in size exclusion chromatography, we have confirmed that the phot1LOV1 domain exists as a dimer in the dark. The D-value and the concentration dependence of the rate indicated that the phot1LOV1 domain associates to form a tetramer (dimerization of the dimer) upon photoexcitation. We also found that the chromophore is released from the binding pocket of the LOV domain when it absorbs two photons within a pulse duration, which occurs in addition to the normal photocycle reaction. On the basis of these results, we discuss the molecular mechanism of the light dependent role of the phot1LOV1 domain.

Signaling mechanisms of LOV domains: new insights from molecular dynamics studies.

Phototropins are one of several classes of photoreceptors used by plants and algae to respond to light. These proteins contain flavin-binding LOV (Light-Oxygen-Voltage) domains that form covalent cysteine-flavin adducts upon exposure to blue light, leading to the enhancement of phototropin kinase activity. Several lines of evidence suggest that adduct formation in the phototropin LOV2 domains leads to the dissociation of an alpha helix (Jα) from these domains as part of the light-induced activation process. However, crystal structures of LOV domains both in the presence and absence of the Jα helix show very few differences between dark and illuminated states, and thus the precise mechanism through which adduct formation triggers helical dissociation remains poorly understood. Using Avena sativa phototropin 1 LOV2 as a model system, we have studied the interactions of the LOV domain core with the Jα helix through a series of equilibrium molecular dynamics simulations. Here we show that conformational transitions of a conserved glutamine residue in the flavin binding pocket are coupled to altered dynamics of the Jα helix both through a shift in dynamics of the main ß-sheet of the LOV domain core and through a secondary pathway involving the N-terminal A'α helix.

Regulatory mechanism of the light-activable allosteric switch LOV-TAP for the control of DNA binding: a computer simulation study.

The spatio-temporal control of gene expression is fundamental to elucidate cell proliferation and deregulation phenomena in living systems. Novel approaches based on light-sensitive multiprotein complexes have recently been devised, showing promising perspectives for the noninvasive and reversible modulation of the DNA-transcriptional activity in vivo. This has lately been demonstrated in a striking way through the generation of the artificial protein construct light-oxygen-voltage (LOV)-tryptophan-activated protein (TAP), in which the LOV-2-Jα photoswitch of phototropin1 from Avena sativa (AsLOV2-Jα) has been ligated to the tryptophan-repressor (TrpR) protein from Escherichia coli. Although tremendous progress has been achieved on the generation of such protein constructs, a detailed understanding of their functioning as opto-genetical tools is still in its infancy. Here, we elucidate the early stages of the light-induced regulatory mechanism of LOV-TAP at the molecular level, using the noninvasive molecular dynamics simulation technique. More specifically, we find that Cys450-FMN-adduct formation in the AsLOV2-Jα-binding pocket after photoexcitation induces the cleavage of the peripheral Jα-helix from the LOV core, causing a change of its polarity and electrostatic attraction of the photoswitch onto the DNA surface. This goes along with the flexibilization through unfolding of a hairpin-like helix-loop-helix region interlinking the AsLOV2-Jα- and TrpR-domains, ultimately enabling the condensation of LOV-TAP onto the DNA surface. By contrast, in the dark state the AsLOV2-Jα photoswitch remains inactive and exerts a repulsive electrostatic force on the DNA surface. This leads to a distortion of the hairpin region, which finally relieves its tension by causing the disruption of LOV-TAP from the DNA.

The amino-terminal helix modulates light-activated conformational changes in AsLOV2.

The mechanism of light-triggered conformational change and signaling in light-oxygen-voltage (LOV) domains remains elusive in spite of extensive investigation and their use in optogenetic studies. The LOV2 domain of Avenasativa phototropin 1 (AsLOV2), a member of the Per-Arnt-Sim (PAS) family, contains a flavin mononucleotide chromophore that forms a covalent bond with a cysteine upon illumination. This event leads to the release of the carboxy-terminal Jα helix, the biological output signal. Using mutational analysis, circular dichroism, and NMR, we find that the largely ignored amino-terminal helix is a control element in AsLOV2's light-activated conformational change. We further identify a direct amino-to-carboxy-terminal "input-output" signaling pathway. These findings provide a framework to rationalize the LOV domain architecture, as well as the signaling mechanisms in both isolated and tandem arrangements of PAS domains. This knowledge can be applied in engineering LOV-based photoswitches, opening up new design strategies and improving existing ones.

Signals of LOV1: a computer simulation study on the wildtype LOV1-domain of Chlamydomonas reinhardtii and its mutants.

Phototropins are photoreceptors regulating the blue-light response in plants and bacteria. They consist of two LOV (light oxygen voltage sensitive) domains each containing a non-covalently bound flavin-mononucleotide (FMN) chromophore, which are connected to a serine/threonine-kinase. Upon illumination, the LOV-domains undergo conformational changes, triggering a signal cascade in the organism through kinase activation. Here, we present results from molecular dynamics simulations in which we investigate the signal transduction pathway of the wildtype LOV1-domain of Chlamydomonas reinhardtii and a methyl-mercaptan (MM) adduct of its Cys57Gly-mutant at the molecular level. In particular, we analyzed the effect of covalent-bond formation between the reactive cysteine Cys57 and the FMN-reaction center, as well as the subsequent charge redistribution, on the spatio-dynamical behavior of the LOV1-domain. We compare the calculation results with experimental data and demonstrate that these adduct state characteristics have an important influence on the response of this photosensor. The light-induced changes implicate primarily an alteration of the surface charge distribution through rearrangement of the highly flexible Cα-, Dα- and Eα-helices including the Glu51-Lys91-salt bridge on the hydrophilic side of the protein domain and a ß-sheet tightening process via coupling of the Aß- and Bß-strands. Our findings confirm the aptitude of the LOV1-domain to function as a dimerization partner, allowing the green alga to adapt its reproduction and growth speed to the environmental conditions.

Modulation of phototropic responsiveness in Arabidopsis through ubiquitination of phototropin 1 by the CUL3-Ring E3 ubiquitin ligase CRL3(NPH3).

Plant phototropism is an adaptive response to changes in light direction, quantity, and quality that results in optimization of photosynthetic light harvesting, as well as water and nutrient acquisition. Though several components of the phototropic signal response pathway have been identified in recent years, including the blue light (BL) receptors phototropin1 (phot1) and phot2, much remains unknown. Here, we show that the phot1-interacting protein NONPHOTOTROPIC HYPOCOTYL3 (NPH3) functions as a substrate adapter in a CULLIN3-based E3 ubiquitin ligase, CRL3(NPH3). Under low-intensity BL, CRL3(NPH3) mediates the mono/multiubiquitination of phot1, likely marking it for clathrin-dependent internalization from the plasma membrane. In high-intensity BL, phot1 is both mono/multi- and polyubiquitinated by CRL3(NPH3), with the latter event targeting phot1 for 26S proteasome-mediated degradation. Polyubiquitination and subsequent degradation of phot1 under high-intensity BL likely represent means of receptor desensitization, while mono/multiubiquitination-stimulated internalization of phot1 may be coupled to BL-induced relocalization of hormone (auxin) transporters.

Modulating LOV domain photodynamics with a residue alteration outside the chromophore binding site.

Phototropins, a class of light-activated protein kinases, are essential for several blue light responses in plants and algae, including phototropism. These proteins contain two internal light, oxygen, and voltage sensitive (LOV) domains, which bind flavin chromophores and undergo a reversible photochemical formation of a cysteinyl-flavin adduct as part of the light sensing process. While the photodynamic properties of such photosensory domains are dictated by interactions between the chromophore and surrounding protein, more distant residues can play a significant role as well. Here we explore the role of the Phe434 residue in the photosensory response of the second LOV domain of Avena sativa phototropin 1 (AsLOV2), a model photochemical system for these LOV domains. Phe434 is more than 6 Å from the FMN chromophore in AsLOV2; nevertheless, an F434Y point mutation is likely to change several structural features of the chromophore binding site, as we demonstrate using molecular dynamics simulations. Transient absorption signals spanning 15 decades in time were compared for wild-type AsLOV2 and the F434Y mutant, showing that the latter has significantly altered photodynamics, including (i) a faster intersystem crossing leading to triplet formation on a nanosecond time scale, (ii) biphasic formation of adduct-state kinetics on the microsecond time scale, and (iii) greatly accelerated ground-state recovery kinetics on a second time scale. We present mechanistic models that link these spectroscopic differences to changes in the configuration of the critical cysteine residue and in the chromophore's accessibility to solvent and oxygen according to MD trajectories and purging experiments. Taken together, these results demonstrate the importance of residues outside the chromophore-binding pocket in modulating LOV domain photodynamics.