Fluorescence Imaging-Based High-Throughput Screening of Fast- and Slow-Cycling LOV Proteins

Kawano, Fuun; Aono, Yuki; Suzuki, Hideyuki; Sato, Moritoshi

doi:10.1371/journal.pone.0082693

Cited by 72 publications

(88 citation statements)

References 26 publications

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“…4). Residue substitutions at this site are known to have large effects on LOV kinetics in VVD and other proteins (18,36). Specifically, an I74V variant of VVD accelerates the photocycle 24-fold; however, it has no effect on solvent accessibility (18).…”

Section: Resultsmentioning

confidence: 99%

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A Native Threonine Coordinates Ordered Water to Tune Light-Oxygen-Voltage (LOV) Domain Photocycle Kinetics and Osmotic Stress Signaling in Trichoderma reesei ENVOY

Lokhandwala

Vega

Hopkins

et al. 2016

Journal of Biological Chemistry

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Light-oxygen-voltage (LOV)3 domain-containing photoreceptors are widely distributed in nature, where they couple blue light absorption to regulation of a diverse array of signal transduction pathways (1). In general, LOV proteins can be divided into two subclasses: 1) short LOV proteins (sLOV) that exist as the isolated LOV domain with short ancillary N-or C-terminal caps (2-4) and 2) modular LOV proteins that couple blue light activation to allosteric regulation of effector domains (1). Although the modular LOV proteins are present in plants, bacteria, and all fungi, the sLOV variety are predominantly found in bacteria and only some fungi (4). Currently, LOV proteins have received widespread attention because of their important roles in regulation of circadian function (3, 5, 6), growth and development (7,8), stress responses (9 -11), and adaptation to and regulation of pathogenicity (12). In addition, their wide ranging utility has led to the development of optogenetic tools that harness their modular design (13,14). Despite substantial research, key questions involving LOV photocycles remain.LOV domain chemistry is characterized by blue light-induced formation of a covalent adduct between a bound flavin cofactor (FMN, FAD, or riboflavin) and a conserved Cys residue in a GXNCRFLQ motif. Concomitant with adduct formation is protonation of the N5 position of the isoalloxazine ring. Current models indicate that signal transduction is coupled to N5 protonation via allosteric regulation of N-or C-terminal effector elements remote from the flavin active site (3,15,16). The covalent adduct is defined by a broad UV-visible absorption band centered at ϳ390 nm (LOV 390 ). Upon return to the dark, the adduct state spontaneously decays to an oxidized flavin (LOV 450 ) on a timescale of seconds to days (17,18). Currently the biological role of the wide range in photocycle lifetimes is unknown; however, several studies have suggested that the range facilitates adaptation to changing levels of light intensity (17,19,20). For these reasons, chemical tuning of the LOV photocycle lifetime through understanding of the adduct decay mechanism has been attempted in several systems (2,17,18,(21)(22)(23)(24).Several lines of reasoning have led to a general mechanism of adduct decay. First, solvent isotope effect experiments indicate that a single proton abstraction event is rate-limiting (17, 18). Second, adduct decay can be catalyzed by the presence of small molecule bases such as imidazole (25). Third, residue substitutions at regions that regulate solvent access to the flavin active site have a substantial effect on LOV photocycle lifetimes (18,26). Combined, these experiments implicate N5 deprotonation as the rate-determining step in adduct decay. Consistent with such a model, mutation of residues that regulate accessibility of small molecules to the N5 position or that tune hydrogen bonding characteristics affect kinetics of LOV proteins (17,18,21,22,24,26,27). Importantly, the natural base responsible for N5 deprotonation remai...

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Section: Resultsmentioning

confidence: 99%

“…Position 101 has been implicated in tuning LOV domain kinetics in numerous systems, but its role in activating water has not been explored (18,31,36,46). Similarly, its effect on signaling mechanisms is not well known.…”

Section: Discussionmentioning

confidence: 99%

A Native Threonine Coordinates Ordered Water to Tune Light-Oxygen-Voltage (LOV) Domain Photocycle Kinetics and Osmotic Stress Signaling in Trichoderma reesei ENVOY

Lokhandwala

Vega

Hopkins

et al. 2016

Journal of Biological Chemistry

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“…The slow photoswitches could be used to avoid some unwanted side effects caused by continuous irradiation, such as phototoxicity and unfocused activation owing to sample drift. Recently, methods to improve switch-off kinetics of blue light photoreceptors have been developed using bacterial or yeast colonies-based high throughput screening systems 28,29 . These screening methods can help to further accelerate or decelerate the switch-off kinetics of the present photoswitches.…”

Section: Discussionmentioning

confidence: 99%

Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins

et al. 2015

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Optogenetic methods take advantage of photoswitches to control the activity of cellular proteins. Here, we completed a multi-directional engineering of the fungal photoreceptor Vivid to develop pairs of distinct photoswitches named Magnets. These new photoswitches were engineered to recognize each other based on the electrostatic interactions, thus preventing homodimerization and enhancing light-induced heterodimerization. Furthermore, we tuned the switch-off kinetics by four orders of magnitude and developed several variants, including those with substantially faster kinetics than any of the other conventional dimerization-based blue spectrum photoswitches. We demonstrate the utility of Magnets as powerful tools that can optogenetically manipulate molecular processes in biological systems.

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“…Given the temperature-dependent change of the lifetime, we predicted that Mpphot perceives temperature using the lifetime of active LOV2 to selectively induce the accumulation response or the cold-avoidance response. To test this possibility, we used an amino acid substitution (V594T) (13) (Fig. S6) that accelerates the speed of thermal reversion fourfold and confirmed that the lifetime of active LOV2…”

Section: Significancementioning

confidence: 99%

Phototropin perceives temperature based on the lifetime of its photoactivated state

Fujii

Tanaka

Konno

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

143

168

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Living organisms detect changes in temperature using thermosensory molecules. However, these molecules and/or their mechanisms for sensing temperature differ among organisms. To identify thermosensory molecules in plants, we investigated chloroplast positioning in response to temperature changes and identified a blue-light photoreceptor, phototropin, that is an essential regulator of chloroplast positioning. Based on the biochemical properties of phototropin during the cellular response to light and temperature changes, we found that phototropin perceives temperature based on the temperature-dependent lifetime of the photoactivated chromophore. Our findings indicate that phototropin perceives both blue light and temperature and uses this information to arrange the chloroplasts for optimal photosynthesis. Because the photoactivated chromophore of many photoreceptors has a temperature-dependent lifetime, a similar temperature-sensing mechanism likely exists in other organisms. Thus, photoreceptors may have the potential to function as thermoreceptors.iving organisms perceive temperature using thermosensory molecules. Various types of thermosensory molecules have been identified in microorganisms, animals, and plants, and these molecules perceive temperature via temperature-dependent intraand intermolecular interactions (1-3). However, these thermosensory molecules and/or their mechanisms for temperature sensing differ among organisms (1).As photosynthetic organelles, chloroplasts change their intracellular position in response to light and temperature (4-7). For example, under low light conditions at 22°C, chloroplasts accumulate at the cell surface (along the periclinal cell wall) to maximize photosynthetic efficiency, in a phenomenon termed the accumulation response (4, 6). However, under the same light conditions, but at a temperature of 5°C, the chloroplasts move to the cell periphery (along the anticlinal cell wall), possibly to avoid the light, in a phenomenon termed the cold-avoidance response or the cold-positioning response (5, 6). Because the cold-avoidance response is temperature dependent (5), the response is likely controlled by a thermosensory molecule. In a previous study, we found that the blue-light (BL) photoreceptor phototropin (phot) is responsible for the cold-avoidance response in the fern Adiantum capillus-veneris, which has three types of phot molecules (phot1, phot2, and neochrome1) (5,8). In this fern, phot2 mediates the cold-avoidance response (5). Therefore, in the present study, we hypothesized that phot is involved in temperature perception and we further analyzed the cold-avoidance response in the liverwort Marchantia polymorpha, which has only a single copy of the PHOT gene (MpPHOT) (9). Results and DiscussionFirst, we examined whether MpPHOT is essential for the coldavoidance response in M. polymorpha. When the wild type (WT), the knockout mutant (Mpphot KO ), and the complementation line (Mpphot/Mpphot KO ) (9) (Fig. S1) were treated with low white light at 5°C for 24 h to induce ...

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Fluorescence Imaging-Based High-Throughput Screening of Fast- and Slow-Cycling LOV Proteins

Cited by 72 publications

References 26 publications

A Native Threonine Coordinates Ordered Water to Tune Light-Oxygen-Voltage (LOV) Domain Photocycle Kinetics and Osmotic Stress Signaling in Trichoderma reesei ENVOY

A Native Threonine Coordinates Ordered Water to Tune Light-Oxygen-Voltage (LOV) Domain Photocycle Kinetics and Osmotic Stress Signaling in Trichoderma reesei ENVOY

Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins

Phototropin perceives temperature based on the lifetime of its photoactivated state

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