Circularly permuted AsLOV2 as an optogenetic module for engineering photoswitchable peptides

Geng, Lequn; Shen, Jiaqi; Wang, Wenjing

doi:10.1039/d1cc02643g

Cited by 10 publications

(8 citation statements)

References 31 publications

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“…To increase the versatility of lightsensing proteins, we have designed cpLOV to cage the Cterminal region of a peptide as well (Figure 6A,B). 6 cpLOV has been successfully applied to cage the TEVcs in the DRD1−SPARK system (Figure 6C−E). 8 We demonstrated the tandem use of both the LOV and cpLOV domains to further improve the light-dependent dynamic range (Figure 6F−H).…”

Section: Protein Switches For Designing Genetically Encoded Tools To ...mentioning

confidence: 99%

“…8 We demonstrated the tandem use of both the LOV and cpLOV domains to further improve the light-dependent dynamic range (Figure 6F−H). 6 cpLOV will significantly expand the toolbox of optogenetic tools by controlling either or both terminal regions of a peptide in a light-dependent manner. A similar work on cpLOV design was reported at a similar time, 69 demonstrating the feasibility and utility of the cpLOV domain.…”

Section: Protein Switches For Designing Genetically Encoded Tools To ...mentioning

confidence: 99%

“…To address the above needs, our lab has designed both chemical-activatable and photoswitchable protein domains for controlling short peptide activity. These protein switches can be used to cage peptides’ agonist activity so that they cannot activate the receptor.…”

Section: Protein Switches For Designing Genetically Encoded Tools To ...mentioning

confidence: 99%

“…However, the LOV domain’s application was limited to caging the N-terminal region of a peptide. To increase the versatility of light-sensing proteins, we have designed cpLOV to cage the C-terminal region of a peptide as well (Figure A,B) …”

Section: Protein Switches For Designing Genetically Encoded Tools To ...mentioning

confidence: 99%

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Protein-Based Tools for Studying Neuromodulation

Wang

2024

ACS Chem. Biol.

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Neuromodulators play crucial roles in regulating neuronal activity and affecting various aspects of brain functions, including learning, memory, cognitive functions, emotional states, and pain modulation. In this Account, we describe our group's efforts in designing sensors and tools for studying neuromodulation. Our lab focuses on developing new classes of integrators that can detect neuromodulators across the whole brain while leaving a mark for further imaging analysis at high spatial resolution. Our lab also designed chemical-and light-dependent protein switches for controlling peptide activity to potentially modulate the endogenous receptors of the neuromodulatory system in order to study the causal effects of selective neuronal pathways.

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Section: Protein Switches For Designing Genetically Encoded Tools To ...mentioning

confidence: 99%

Section: Protein Switches For Designing Genetically Encoded Tools To ...mentioning

confidence: 99%

Section: Protein Switches For Designing Genetically Encoded Tools To ...mentioning

confidence: 99%

Section: Protein Switches For Designing Genetically Encoded Tools To ...mentioning

confidence: 99%

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Protein-Based Tools for Studying Neuromodulation

Wang

2024

ACS Chem. Biol.

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“…This construct was first utilized in yeast cells 17 and has now been adapted to allow expression in mammalian cells (HEK293 cells and neurons), using cell-type specific promoters. The 4E-BP2 fragment in cLIPS is comprised of the wild-type 4E-BP2 sequence from position 51-85 with mutated phosphorylation sites (S65A and T70A), whereas the photoswitchable domain in cLIPS is a circularly permuted version of AsLOV2, the second light-oxygen-voltage domain of Avena sativa phototropin 1 [17][18][19][20][21][22] , containing a point mutation V92I that slows the photocycle and thereby increases the light sensitivity 17 . To enable visualization of cells expressing Opto4E-BP, a fluorescence reporter mCherry was included in the construct and separated from Opto4E-BP with self-cleaving peptide P2A allowing ribosome reinitiation 23,24 (Fig.…”

Section: An Optogenetic Tool For Cell-type Specific Protein Synthesis...mentioning

confidence: 99%

Opto4E-BP, an optogenetic tool for inducible, reversible, and cell type-specific inhibition of translation initiation

Alapin,

Mohamed,

Shrestha

et al. 2023

Preprint

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The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is one of the primary triggers for initiating cap-dependent translation. Amongst its functions, mTORC1 phosphorylates eIF4E-binding proteins (4E-BPs), which prevents them from binding to eIF4E and thereby enables translation initiation. mTORC1 signaling is required for multiple forms of protein synthesis- dependent synaptic plasticity and various forms of long-term memory (LTM), including associative threat memory. However, the approaches used thus far to target mTORC1 and its effectors, such as pharmacological inhibitors or genetic knockouts, lack fine spatial and temporal control. The development of a conditional and inducible eIF4E knockdown mouse line partially solved the issue of spatial control, but still lacked optimal temporal control to study memory consolidation. Here, we have designed a novel optogenetic tool (Opto4E-BP) for cell type-specific, light-dependent regulation of eIF4E in the brain. We show that light-activation of Opto4E-BP decreases protein synthesis in HEK cells and primary mouse neurons. In situ, light-activation of Opto4E-BP in excitatory neurons decreased protein synthesis in acute amygdala slices. Finally, light activation of Opto4E-BP in principal excitatory neurons in the lateral amygdala (LA) of mice after training blocked the consolidation of LTM. The development of this novel optogenetic tool to modulate eIF4E-dependent translation with spatiotemporal precision will permit future studies to unravel the complex relationship between protein synthesis and the consolidation of LTM.

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Rational Design of a Circularly Permuted Flavin‐Based Fluorescent Protein

Anderson,

Xie,

Chacko

et al. 2024

ChemBioChem

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Flavin‐based fluorescent proteins are oxygen‐independent reporters that hold great promise for imaging anaerobic and hypoxic biological systems. In this study, we explored the feasibility of applying circular permutation, a valuable method for the creation of fluorescent sensors, to flavin‐based fluorescent proteins. We used rational design and structural data to identify a suitable location for circular permutation in iLOV, a flavin‐based reporter derived from A. thaliana. However, relocating the N‐ and C‐termini to this position resulted in a significant reduction in fluorescence. This loss of fluorescence was reversible, however, by fusing dimerizing coiled coils at the new N‐ and C‐termini to compensate for the increase in local chain entropy. Additionally, by inserting protease cleavage sites in circularly permuted iLOV, we developed two protease sensors and demonstrated their application in mammalian cells. In summary, our work establishes the first approach to engineer circularly permuted FbFPs optimized for high fluorescence and further showcases the utility of circularly permuted FbFPs to serve as a scaffold for sensor engineering.

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Circularly permuted AsLOV2 as an optogenetic module for engineering photoswitchable peptides

Abstract: A circularly permuted AsLOV2 domain has been designed to photocontrol peptides by caging their C-terminus. This photoswitch provides a new module for engineering optogenetic tools and a general approach to lower the background of LOV-based tools.

Cited by 10 publications

References 31 publications

Protein-Based Tools for Studying Neuromodulation

Protein-Based Tools for Studying Neuromodulation

Opto4E-BP, an optogenetic tool for inducible, reversible, and cell type-specific inhibition of translation initiation

Rational Design of a Circularly Permuted Flavin‐Based Fluorescent Protein

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