Guidelines for Photoreceptor Engineering

Ziegler, Thea; Schumacher, Charlotte Helene; Möglich, Andreas

doi:10.1007/978-1-4939-3512-3_27

Cited by 6 publications

(4 citation statements)

References 64 publications

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“…Importantly, the N and C termini of LOV2 are oriented towards the same direction making it suitable to insert into other proteins [41][42][43], and in the case of PKM2, into the HLH region. Furthermore, LOV2 is suitable for use in mammalian cells [44], which tolerate blue light well and do not need exogenous cofactors because they produce FMN from dietary vitamin B. Altogether, we concluded that LOV2 is a suitable domain to insert into PKM2 and potentially regulate its activity and for subsequent use in mammalian cells.…”

Section: Molecular Dynamics Simulations Identify Conformational Changmentioning

confidence: 73%

An engineered photoswitchable mammalian pyruvate kinase

et al. 2017

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Changes in allosteric regulation of glycolytic enzymes have been linked to metabolic reprogramming involved in cancer. Remarkably, allosteric mechanisms control enzyme function at significantly shorter time‐scales compared to the long‐term effects of metabolic reprogramming on cell proliferation. It remains unclear if and how the speed and reversibility afforded by rapid allosteric control of metabolic enzymes is important for cell proliferation. Tools that allow specific, dynamic modulation of enzymatic activities in mammalian cells would help address this question. Towards this goal, we have used molecular dynamics simulations to guide the design of mPKM2 internal light/oxygen/voltage‐sensitive domain 2 (LOV2) fusion at position D24 (PiL[D24]), an engineered pyruvate kinase M2 (PKM2) variant that harbours an insertion of the light‐sensing LOV2 domain from Avena Sativa within a region implicated in allosteric regulation by fructose 1,6‐bisphosphate (FBP). The LOV2 photoreaction is preserved in the PiL[D24] chimera and causes secondary structure changes that are associated with a 30% decrease in the K m of the enzyme for phosphoenolpyruvate resulting in increased pyruvate kinase activity after light exposure. Importantly, this change in activity is reversible upon light withdrawal. Expression of PiL[D24] in cells leads to light‐induced increase in labelling of pyruvate from glucose. PiL[D24] therefore could provide a means to modulate cellular glucose metabolism in a remote manner and paves the way for studying the importance of rapid allosteric phenomena in the regulation of metabolism and enzyme control.

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Section: Molecular Dynamics Simulations Identify Conformational Changmentioning

confidence: 73%

An engineered photoswitchable mammalian pyruvate kinase

et al. 2017

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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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“…Engineering of optogenetic tools requires not only successful control of an effector domain with a photoreceptor but also optimizations to enhance the tool performance. 3,4 An often used photoreceptor for development of optogenetic tools is the light oxygen voltage (LOV) domain 2 of plant phototropins. 5 This photoreceptor binds to the flavin mononucleotide (FMN), which is primarily responsible for light perception.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Modern methods of genetic engineering facilitate implementation of a tool into the genome of host cells. , Optogenetic tools have a dual functionality; a photoreceptor part perceives the light signal, and an effector domain induces a specific response. Engineering of optogenetic tools requires not only successful control of an effector domain with a photoreceptor but also optimizations to enhance the tool performance. , …”

Section: Introductionmentioning

confidence: 99%

An Optogenetic Toolbox for Synergistic Regulation of Protein Abundance

et al. 2021

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Optogenetic tools have been proven to be useful in regulating cellular processes via an external signal. Light can be applied with high spatial and temporal precision as well as easily modulated in quantity and quality. Natural photoreceptors of the light oxygen voltage (LOV) domain family have been characterized in depth, especially the LOV2 domain of Avena sativa (As) phototropin 1 and its derivatives. Information on the behavior of LOV2 variants with changes in the photocycle or the light response has been recorded. Here, we applied well-described photocycle mutations on the AsLOV2 domain of a photosensitive transcription factor (psTF) as well as its variant that is part of the photosensitive degron (psd) psd3 in Saccharomyces cerevisiae. In vivo and in vitro measurements revealed that each photoreceptor component of the light-sensitive transcription factor and the psd3 module can be modulated in its light sensitivity by mutations that are known to prolong or shorten the dark-reversion time of AsLOV2. Yet, only two of the mutations showed differences in the in vivo behavior in the context of the psd3 module. For the AsLOV2 domain in the context of the psTF, we observed different characteristics for all four variants. Molecular dynamics simulations showed distinct influences of the shortened Jα helix and the V416L mutation in the context of the psd3 photoreceptor. In conclusion, we demonstrated the tunability of two optogenetic tools with a set of mutations that affect the photocycle of the inherent photoreceptors. As these optogenetic tools are concurrent in their action, pleiotropic effects on target protein abundance are achievable with the simultaneous action of the diverse photoreceptor variants.

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Guidelines for Photoreceptor Engineering

Cited by 6 publications

References 64 publications

An engineered photoswitchable mammalian pyruvate kinase

An engineered photoswitchable mammalian pyruvate kinase

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

An Optogenetic Toolbox for Synergistic Regulation of Protein Abundance

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