Rationally improving LOV domain–based photoswitches

Strickland, Devin; Yao, Xiaolan; Gawlak, Grzegorz; Rosen, Michael K.; Gardner, Kevin H.; Sosnick, Tobin R.

doi:10.1038/nmeth.1473

Cited by 179 publications

(258 citation statements)

References 28 publications

(34 reference statements)

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“…Previous studies suggest that a major source of dark state "leakiness" comes from transient undocking and unfolding of AsLOV2's Jα-helix, and mutations that increase the intrinsic stability of the Jα helix have proven successful in reducing darkstate activity of light-activated AsLOV2 switches (9,19). We sought to extend this approach and use molecular modeling combined with high-throughput screening to find mutations throughout the domain that stabilize the docked state of the Jα-helix.…”

Section: Resultsmentioning

confidence: 99%

Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins

Guntas¹,

Hallett²,

Zimmerman³

et al. 2014

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

569

719

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The discovery of light-inducible protein-protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through lightmediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.optogenetic tool | PER-ARNT-SIM domain | computational library | phage display | Rosetta molecular modeling suite

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Section: Resultsmentioning

confidence: 99%

Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins

Guntas¹,

Hallett²,

Zimmerman³

et al. 2014

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

569

719

View full text Add to dashboard Cite

show abstract

“…We chose the AsLOV2-PAH1b construct for all subsequent experiments, as it was the one showing the best effect in the open conformation. To further improve the stability of the chimera in the dark, we introduced the I532A point mutation in the AsLOV2 sequence, known to increase Jα-helix stability (36). The introduction of the second mutation completely abolished the residual activity of the dark construct on the exogenously expressed RE1-SV40 promoter (Fig.…”

Section: Resultsmentioning

confidence: 99%

Regulation of neural gene transcription by optogenetic inhibition of the RE1-silencing transcription factor

Paonessa

Criscuolo

Sacchetti

et al. 2015

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

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Optogenetics provides new ways to activate gene transcription; however, no attempts have been made as yet to modulate mammalian transcription factors. We report the light-mediated regulation of the repressor element 1 (RE1)-silencing transcription factor (REST), a master regulator of neural genes. To tune REST activity, we selected two protein domains that impair REST-DNA binding or recruitment of the cofactor mSin3a. Computational modeling guided the fusion of the inhibitory domains to the light-sensitive Avena sativa lightoxygen-voltage-sensing (LOV) 2-phototrophin 1 (AsLOV2). By expressing AsLOV2 chimeras in Neuro2a cells, we achieved light-dependent modulation of REST target genes that was associated with an improved neural differentiation. In primary neurons, light-mediated REST inhibition increased Na + -channel 1.2 and brain-derived neurotrophic factor transcription and boosted Na + currents and neuronal firing. This optogenetic approach allows the coordinated expression of a cluster of genes impinging on neuronal activity, providing a tool for studying neuronal physiology and correcting gene expression changes taking place in brain diseases.

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“…We anticipate that the elucidation of the AsLOV2-Jα signal-transduction pathway at a molecular level presented in this work will provide new perspectives for the creation of novel fusion proteins in combination with the AsLOV2-Jα photosensor. As demonstrated on the example of its asparagine mutant AsLOV2-Jα-Gln1029Asn, this knowledge can be used to engineer novel fusion proteins with optimized light-induced response, which might open new avenues for allosteric control of protein activity in living cells [22][23][24][25] .…”

Section: Discussionmentioning

confidence: 99%

Mechanism of signal transduction of the LOV2-Jα photosensor from Avena sativa

2010

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Fusion proteins containing blue-light-activable protein domains possess great potential as molecular switches in cell signalling. This has recently been impressively demonstrated by connecting the light oxygen voltage LoV2-Jα-protein domain of A. sativa (AsLoV2-Jα) with the Rac1-GTPase, responsible for regulating the morphology and motility of metazoan cells. However, a target-oriented development of fusion proteins in conjunction with this photosensor is still very challenging, because a detailed understanding of its signal transduction pathway on a molecular level is still lacking. Here, we show through molecular dynamics simulation that, after formation of the cysteinyl-flavin mononucleotide (Fmn) adduct, the signalling pathway begins with a rotational reorientation of the residue glutamine 1029 adjacent to the Fmn chromophore, transmitting stress through the Iβ strand towards the LoV2-Jα interface. This then results in the breakage of two H-bonds, namely, glutamic acid 1034-Gln995 and aspartic acid (Asp) 1056-Gln1013, at opposite sides of the interface between the Jα helix and the LoV2 domain, ultimately leading to a disruption of Jα helix from the LoV2 core.

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Rationally improving LOV domain–based photoswitches

Cited by 179 publications

References 28 publications

Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins

Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins

Regulation of neural gene transcription by optogenetic inhibition of the RE1-silencing transcription factor

Mechanism of signal transduction of the LOV2-Jα photosensor from Avena sativa

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