Detection of Incorporation of <i>p</i>-Coumaric Acid into Photoactive Yellow Protein Variants <i>in Vivo</i>

Brechun, Katherine E.; Zhen, Danlin; Jaikaran, Anna S. I.; Borisenko, Vitali; Kumauchi, Masato; Hoff, Wouter D.; Arndt, Katja M.; Woolley, G. Andrew

doi:10.1021/acs.biochem.9b00279

Cited by 6 publications

(4 citation statements)

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“…Finally, an M100E mutation was made to slow the thermal relaxation rate of the PYP light‐state and thereby decrease the light intensity required for photoswitching and avoid potential light toxicity in vivo 32,33 . Unlike the LOV domain, PYP uses a p ‐coumaric acid chromophore that must usually be added to cells 34 . However, while unfolding of LOV is not generally reversible, 35 covalent linkage of the chromophore in PYP means that protein folding is fully reversible without chromophore loss.…”

Section: Resultsmentioning

confidence: 99%

“…32,33 Unlike the LOV domain, PYP uses a pcoumaric acid chromophore that must usually be added to cells. 34 However, while unfolding of LOV is not generally reversible, 35 covalent linkage of the chromophore in PYP means that protein folding is fully reversible without chromophore loss. PYP has been used as the light switching domain in optogenetic tools in vitro, in live cells, and in vivo.…”

Section: Protein Designmentioning

confidence: 99%

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Structure‐based design of a photoswitchable affibody scaffold

et al. 2021

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Photo‐control of affinity reagents offers a general approach for high‐resolution spatiotemporal control of diverse molecular processes. In an effort to develop general design principles for a photo‐controlled affinity reagent, we took a structure‐based approach to the design of a photoswitchable Z‐domain, among the simplest of affinity reagent scaffolds. A chimera, designated Z‐PYP, of photoactive yellow protein (PYP) and the Z‐domain, was designed based on the concept of mutually exclusive folding. NMR analysis indicated that, in the dark, the PYP domain of the chimera was folded, and the Z‐domain was unfolded. Blue light caused loss of structure in PYP and a two‐ to sixfold change in the apparent affinity of Z‐PYP for its target as determined using size exclusion chromatography, UV‐Vis based assays, and enyzme‐linked immunosorbent assay (ELISA). A thermodynamic model indicated that mutations to decrease Z‐domain folding energy would alter target affinity without loss of switching. This prediction was confirmed experimentally with a double alanine mutant in helix 3 of the Z‐domain of the chimera (Z‐PYP‐AA) showing >30‐fold lower dark‐state binding and no loss in switching. The effect of decreased dark‐state binding affinity was tested in a two‐hybrid transcriptional control format and enabled pronounced light/dark differences in yeast growth in vivo. Finally, the design was transferable to the αZ‐Taq affibody enabling tunable light‐dependent binding both in vitro and in vivo to the Z‐Taq target. This system thus provides a framework for the focused development of light switchable affibodies for a range of targets.

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Section: Resultsmentioning

confidence: 99%

Section: Protein Designmentioning

confidence: 99%

Structure‐based design of a photoswitchable affibody scaffold

et al. 2021

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“…In optogenetics, this protein can be used as a tunable optical switch because blue light triggers trans to cis isomerization of the chromophore and production of a light state with altered conformational dynamics. Based on this property, PYP has been used as switchable protein in optogenetics and it is usually linked to effector domains resulting in soluble, well-behaved constructs [44,45]. This feasibility to form specific constructs has been used to design structure-based photoswitchable affibody scaffolds for applications in immunochemistry, particularly in the use of antibodies using the tunable properties of the PYP-Z-domain with a light-dependent binding in vitro and in vivo [46].…”

Section: Introductionmentioning

confidence: 99%

A Short and Practical Overview on Light-Sensing Proteins, Optogenetics, and Fluorescent Biomolecules inside Biomorphs Used as Optical Sensors

Galindo-García,

Vanegas-Reza,

Arreguín-Espinosa

et al. 2023

Crystals

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In this contribution, we describe a brief overview of the role of different light-signaling proteins in different biochemical processes (mostly in plants) along the electromagnetic spectrum. We also revise, in terms of perspectives, the applications of all these proteins to optogenetics as a new emerging field of research. In the second part, we present some case studies: First, we used two fluorescent proteins showing an optical response in the green- and red-light wavelengths both isolated from marines’ organisms, which were incorporated as light sensors into the silico-carbonate of Ca, Ba, and Sr (usually called biomorphs). The second case study consisted in incorporating phototropins from a plant (Arabidopsis thaliana) into the synthesis of biomorphs. Finally, the last part analyses the influence of these three proteins on the shape and structure in the synthesis of silico-carbonates of calcium, barium, and strontium as optical sensors, in order to detect the location of these biomolecules inside these self-assembly crystalline materials called biomorphs.

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“…However, their utility is hampered by lack of a generalizable photodomain insertion site with predicable effects on binding 18,19 , variable levels of baseline binding and light-inducibility across targets [18][19][20] , or the need for exogenous chromophore supply. 20,21 Finally, while the reported tools can in principle regulate endogenous proteins, few endogenous targets have so far been studied using SBDs with photoinducible binding. 17…”

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confidence: 99%

Photoswitchable binders enable temporal dissection of endogenous protein function

Westberg,

Song,

Duong

et al. 2023

Preprint

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General methods for spatiotemporal control of specific endogenous proteins would be broadly useful for probing protein function in living cells. Synthetic protein binders that bind and inhibit endogenous protein targets can be obtained from nanobodies, designed ankyrin repeat proteins (DARPins), and other small protein scaffolds, but generalizable methods to control their binding activity are lacking. Here, we report robust single-chain photoswitchable DARPins (psDARPins) for bidirectional optical control of endogenous proteins. We created topological variants of the DARPin scaffold by computer-aided design so fusion of photodissociable dimeric Dronpa (pdDronpa) results in occlusion of target binding at baseline. Cyan light induces pdDronpa dissociation to expose the binding surface (paratope), while violet light restores pdDronpa dimerization and paratope caging. Since the DARPin redesign leaves the paratope intact, the approach was easily applied to existing DARPins for GFP, ERK, and Ras, as demonstrated by relocalizing GFP-family proteins and inhibiting endogenous ERK and Ras with optical control. Finally, a Ras-targeted psDARPin was used to determine that, following EGF-activation of EGFR, Ras is required for sustained EGFR to ERK signaling. In summary, psDARPins provide a generalizable strategy for precise spatiotemporal dissection of endogenous protein function.

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Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo

Cited by 6 publications

References 61 publications

Structure‐based design of a photoswitchable affibody scaffold

Structure‐based design of a photoswitchable affibody scaffold

A Short and Practical Overview on Light-Sensing Proteins, Optogenetics, and Fluorescent Biomolecules inside Biomorphs Used as Optical Sensors

Photoswitchable binders enable temporal dissection of endogenous protein function

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