Photoreceptor engineering

Ziegler, Thea; MÃ¶glich, Andreas

doi:10.3389/fmolb.2015.00030

Cited by 115 publications

(153 citation statements)

References 166 publications

(326 reference statements)

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“…Photoreceptors comprise photosensor modules which absorb incident light and effector modules which possess biological activity and elicit downstream physiological adaptations (1). This modular, genetically encodable architecture lends itself to the engineering of novel photoreceptors via recombination of photosensors and effectors (2). Engineered photoreceptors not only serve as light-regulated actuators in optogenetics, but also provide mechanistic insight into principles of signal transduction.…”

Section: Introductionmentioning

confidence: 99%

“…Engineered photoreceptors not only serve as light-regulated actuators in optogenetics, but also provide mechanistic insight into principles of signal transduction. The two most versatile and successful photoreceptor engineering strategies to date (2) rely on light-regulated order-disorder transitions (3) and light-regulated association reactions (4), respectively. Although numerous effector activities have thus been subjected under light control, no generally applicable route towards creating photoactivatable variants of arbitrary effectors yet exists, especially in case the effector is monomeric and no natural regulatory mechanism is known (2).…”

Section: Introductionmentioning

confidence: 99%

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Engineering of temperature- and light-switchable Cas9 variants

Richter

Fonfara

Bouazza

et al. 2013

Nucleic Acids Research

114

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Sensory photoreceptors have enabled non-invasive and spatiotemporal control of numerous biological processes. Photoreceptor engineering has expanded the repertoire beyond natural receptors, but to date no generally applicable strategy exists towards constructing light-regulated protein actuators of arbitrary function. We hence explored whether the homodimeric Rhodobacter sphaeroides light-oxygen-voltage (LOV) domain (RsLOV) that dissociates upon blue-light exposure can confer light sensitivity onto effector proteins, via a mechanism of light-induced functional site release. We chose the RNA-guided programmable DNA endonuclease Cas9 as proof-of-principle effector, and constructed a comprehensive library of RsLOV inserted throughout the Cas9 protein. Screening with a high-throughput assay based on transcriptional repression in Escherichia coli yielded paRC9, a moderately light-activatable variant. As domain insertion can lead to protein destabilization, we also screened the library for temperature-sensitive variants and isolated tsRC9, a variant with robust activity at 29°C but negligible activity at 37°C. Biochemical assays confirmed temperature-dependent DNA cleavage and binding for tsRC9, but indicated that the light sensitivity of paRC9 is specific to the cellular setting. Using tsRC9, the first temperature-sensitive Cas9 variant, we demonstrate temperature-dependent transcriptional control over ectopic and endogenous genetic loci. Taken together, RsLOV can confer light sensitivity onto an unrelated effector; unexpectedly, the same LOV domain can also impart strong temperature sensitivity.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Engineering of temperature- and light-switchable Cas9 variants

Richter

Fonfara

Bouazza

et al. 2013

Nucleic Acids Research

114

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“…Phototropins, cryptochromes, and blue light using flavin adenine dinucleotide (BLUF) proteins each bind a flavin derivative as a chromophore that undergoes covalent bond formation with a cysteine residue, electron transfer (ET), and proton-coupled electron transfer (PCET), respectively, during the photocycle. Understanding the fundamental mechanisms of naturally occurring photoreceptor proteins is important for engineering novel systems that use light as a tool to achieve noninvasive control of biological processes with high spatiotemporal resolution (5)(6)(7).…”

mentioning

confidence: 99%

Role of active site conformational changes in photocycle activation of the AppA BLUF photoreceptor

Goyal

Hammes‐Schiffer

2017

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

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Blue light using flavin adenine dinucleotide (BLUF) proteins are essential for the light regulation of a variety of physiologically important processes and serve as a prototype for photoinduced proton-coupled electron transfer (PCET). Free-energy simulations elucidate the active site conformations in the AppA (activation of photopigment and puc expression) BLUF domain before and following photoexcitation. The free-energy profile for interconversion between conformations with either Trp104 or Met106 closer to the flavin, denoted Trp in /Met out and Trp out /Met in , reveals that both conformations are sampled on the ground state, with the former thermodynamically favorable by ∼3 kcal/mol. These results are consistent with the experimental observation of both conformations. To analyze the proton relay from Tyr21 to the flavin via Gln63, the free-energy profiles for Gln63 rotation were calculated on the ground state, the locally excited state of the flavin, and the charge-transfer state associated with electron transfer from Tyr21 to the flavin. For the Trp in /Met out conformation, the hydrogenbonding pattern conducive to the proton relay is not thermodynamically favorable on the ground state but becomes more favorable, corresponding to approximately half of the configurations sampled, on the locally excited state. The calculated energy gaps between the locally excited and charge-transfer states suggest that electron transfer from Tyr21 to the flavin is more facile for configurations conducive to proton transfer. When the active site conformation is not conducive to PCET from Tyr21, Trp104 can directly compete with Tyr21 for electron transfer to the flavin through a nonproductive pathway, impeding the signaling efficiency.photoreceptor | BLUF | proton transfer | electron transfer | hydrogen bonding

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“…105 Since the active form is less stable than the inactive form, a thermally driven spontaneous process called dark reversion slowly (milliseconds to many hours) recovers the inactive form, which closes the photocycle. 104 In addition to the dark reversion, photochromic photoreceptors like…”

Section: -104mentioning

confidence: 99%

Computational Studies of Photobiological Keto-Enol Reactions and Chromophores

Falklöf¹

2015

Linköping Studies in Science and Technology. Dissertations

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This thesis presents computational chemistry studies of keto-enol reactions and chromophores of photobiological significance.The first part of the thesis is concerned with two protein-bound chromophores that, depending on the chemical conditions, can exist in a number of different ketonic and enolic forms. The first chromophore is astaxanthin, which occurs in the protein complex responsible for the deep-blue color of lobster carapace. By investigating how different forms of astaxanthin absorb UV-vis radiation of different wavelengths, a model is presented that explains the origin of the dramatic color change from deep-blue to red upon cooking of live lobsters.The second chromophore is the oxyluciferin light emitter of fireflies, which is formed in the catalytic center of the enzyme firefly luciferase. To date, there is no consensus regarding which of the possible ketonic and enolic forms is the key contributor to the light emission. In the thesis, the intrinsic tendency of oxyluciferin to prefer one particular form over other possible forms is established through calculation of keto-enol and acid-base excited-state equilibrium constants in aqueous solution.The second part of the thesis is concerned with two families of biological photoreceptors: the blue-light-absorbing LOV-domain proteins and the red-lightabsorbing phytochromes. Based on the ambient light environment, these proteins regulate physiological and developmental processes by switching between inactive and active conformations. In both families, the conversion of the inactive into the active conformation is triggered by a chemical reaction of the respective chromophore.The LOV-domain proteins bind a flavin chromophore and regulate processes such as chloroplast relocation and phototropism in plants. An important step in the activation of these photoreceptors is a singlet-triplet transition between two electronically excited states of the flavin chromophore. In the thesis, this transition is used as a prototype example for illustrating, for the first time, the ability of first-principles methods to calculate rate constants of inter-excited state phosphorescence events.Phytochromes, in turn, bind bilin chromophores and are active in the regulation of processes like seed germination and flowering time in plants. Following two systematic studies identifying the best way to model the UV-vis absorption iii iv and fluorescence spectra of these photoreceptors, it is demonstrated that steric interactions between the chromophore and the apoprotein play a decisive role for how phytochromes are activated by light. Populärvetenskaplig sammanfattningSolljusär en förutsättning för liv på jorden. Växter använder solljus som energikälla för att kunna bilda näring till sig själva ochäven syre till andra organismer. Iögonen hos människor och djur finns specialdesignade celler som omvandlar ljus till elektriska impulser, som sedan tolkas till en bild av syncentrat i hjärnan. Djur har utvecklat mekanismer för att med hjälp av ljus kunna kamouflera sig, locka till s...

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Photoreceptor engineering

Cited by 115 publications

References 166 publications

Engineering of temperature- and light-switchable Cas9 variants

Engineering of temperature- and light-switchable Cas9 variants

Role of active site conformational changes in photocycle activation of the AppA BLUF photoreceptor

Computational Studies of Photobiological Keto-Enol Reactions and Chromophores

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