Light-activated DNA binding in a designed allosteric protein

Strickland, Devin; Moffat, Keith; Sosnick, Tobin R.

doi:10.1073/pnas.0709610105

Cited by 280 publications

(277 citation statements)

References 47 publications

(56 reference statements)

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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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Section: Discussionmentioning

confidence: 99%

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

2010

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“…2A). The involvement of α-helices in mediating intramolecular signal transduction is not unique to bacteriophytochromes, and other researchers have used such helices for heterologous domain replacements (43)(44)(45)(46)(47)(48)(49).…”

Section: Discussionmentioning

confidence: 99%

Engineering adenylate cyclases regulated by near-infrared window light

Ryu

Kang

Nelson

et al. 2014

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

107

114

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Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochromebased optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset.phytochrome | protein engineering | adenylyl cyclase | cAMP | locomotion

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“…AsLOV2 comprises an inner flavinbinding domain and a C-terminal α-helix (Jα) folded in the dark. On blue light (450-470 nm) stimulation, photon absorption leads to major conformational changes that result in the unwinding of the Jα helix and exposure of the C-terminal fused domain (23,24).…”

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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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Light-activated DNA binding in a designed allosteric protein

Cited by 280 publications

References 47 publications

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

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

Engineering adenylate cyclases regulated by near-infrared window light

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

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