Source 1primary paper2004Proceedings of SPIE, the International Society for Optical Engineering/Proceedings of SPIE
Toolkit/LOV2 domain of Avena sativa phototropin 1
LOV2 domain of Avena sativa phototropin 1
Also known as: Avena sativa phototropin 1 LOV2 domain, LOV2, LOV2 derived from Avena sativa (oat) phot1, LOV2 domain
Taxonomy: Mechanism Branch / Component. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
The LOV2 domain of Avena sativa phototropin 1 is a blue-light-responsive protein domain that uses an FMN-dependent photocycle to reversibly switch between dark and lit states through formation and decay of a flavin-cysteinyl adduct. It has been repurposed as a modular photoswitch to control nuclear import/export motif exposure and to generate light-dependent inhibitory peptides.
Usefulness & Problems
Why this is useful
This domain is useful as a genetically encoded photoswitch for reversible optical control of protein localization and peptide-mediated inhibition in living cells. The cited engineering work shows that embedding nuclear localization or export motifs in the LOV2 C-terminal helix enables light-dependent nucleocytoplasmic trafficking, and that LOV2 can confer light dependence on kinase inhibitor analogues.
Problem solved
It addresses the problem of imposing reversible, light-dependent control over protein function and intracellular localization using a compact protein domain. Specifically, the literature describes solutions for optically controlling nucleocytoplasmic distribution and for making inhibitory peptides active in a light-dependent manner.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Component: A low-level protein part used inside a larger architecture that realizes a mechanism.
Mechanisms
light-dependent control of nucleocytoplasmic traffickinglight-gated steric uncaging of embedded sequence motifslight-induced reversible flavin-cysteinyl adduct formationthermal dark-state relaxationTarget processes
recombinationselectionsignalingInput: Light
Implementation Constraints
The domain is FMN-dependent and its light response is based on a conserved cysteine that forms a covalent adduct with the flavin upon irradiation. Engineering examples used embedding of nuclear import or export motifs into the LOV2 C-terminal helix and tuning by hinge-loop mutagenesis. One screening workflow used Escherichia coli colonies expressing mutagenized LOV2 variants to identify altered blue-light photochemical reactivity.
The supplied evidence does not provide a general quantitative benchmark for activation wavelength, switching kinetics in engineered cellular contexts, or performance across many target proteins. Some mechanistic measurements were made under low-temperature spectroscopic conditions, which may not directly report behavior in standard live-cell applications. Practical performance details for the LOV2-based kinase inhibitor analogues are not extensively described in the provided evidence.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Source 2primary paper2007Biochemistry
Source 3primary paper2018ACS Synthetic Biology
Source 4primary paper2014ACS Synthetic Biology
Ranked Claims
LANS and LINX were engineered by embedding nuclear import or export sequence motifs into the C-terminal helix of the LOV2 domain of Avena sativa phototropin 1 to enable light-dependent trafficking of a target protein into and out of the nucleus.
We previously engineered two optogenetic systems, the light activated nuclear shuttle (LANS) and the light-inducible nuclear exporter (LINX), by embedding nuclear import or export sequence motifs into the C-terminal helix of the light-responsive LOV2 domain of Avena sativa phototropin 1, thus enabling light-dependent trafficking of a target protein into and out of the nucleus.
Hinge-loop mutations in LOV2 were identified that favorably shift the dynamic range of the LANS and LINX photoswitches.
Here, we identify hinge-loop mutations that favorably shift the dynamic range (the ratio of the on- to off-target subcellular accumulation) of the LANS and LINX photoswitches.
dynamic range favorably shifted
The authors developed LOV2-based analogues of kinase inhibitors whose activity is light dependent.
Using the photoresponse of the LOV2 domain of Avena sativa phototropin 1, we developed analogues of kinase inhibitors whose activity is light dependent.
The LOV domain forms a flavin-cysteinyl adduct upon irradiation and thermally relaxes back to the ground state in the dark, functioning as a molecular switch controlling receptor kinase activity.
Irradiation of the LOV domain results in the formation of a flavin-cysteinyl adduct (LOV390) which thermally relaxes back to the ground state in the dark, effectively completing a photocycle that serves as a molecular switch to control receptor kinase activity.
Decreasing the size of the isoleucine side chain accelerates adduct decay, indicating that steric interactions of this side chain stabilize the C-S cysteinyl adduct.
A kinetic acceleration trend was observed for adduct decay by decreasing the size of the isoleucine side chain. Our findings therefore indicate that the steric nature of this amino acid side chain contributes to stabilization of the C-S cysteinyl adduct.
A single Ile-to-Val substitution in the LOV2 domain caused slower LOV390 formation and adduct decay about one order of magnitude faster than wild type.
One variant showed slower rates of LOV390 formation but exhibited adduct decay times 1 order of magnitude faster than wild type. A single Ile --> Val substitution was responsible for the effects observed
adduct decay time change versus wild type 1 order of magnitude faster
No spectral shifting of flavin emission was observed in the LOV2 domain or in FMN in aqueous solution.
No spectral shifting of the flavin emission was observed in the LOV2 domain nor in FMN in aqueous solution.
Phototropins base their light-dependent action on the reversible formation of a covalent bond between an FMN cofactor and a conserved cysteine residue in LOV domains.
The phototropins are plant blue-light receptors that base their light-dependent action on the reversible formation of a covalent bond between a flavin mononucleotide (FMN) cofactor and a conserved cysteine residue in light, oxygen or voltage (LOV) domains.
A pronounced phosphorescence emission around 600 nm was observed in the LOV2 domain between 77 and 120 K, placing the flavin triplet state at 16900 cm^-1.
A pronounced phosphorescence emission around 600 nm was observed in the LOV2 domain between 77 and 120 K, allowing for an accurate positioning of the flavin triplet state in the LOV2 domain at 16900 cm-1.
flavin triplet state position 16900 cm^-1phosphorescence emission maximum 600 nm
The fluorescence quantum yield of the flavin cofactor in the LOV2 domain increased from 0.13 at room temperature to 0.41 at 77 K.
The fluorescence quantum yield of the flavin cofactor increased from 0.13 to 0.41 upon cooling the sample from room temperature to 77 K.
fluorescence quantum yield 0.13fluorescence quantum yield 0.41
Time-resolved fluorescence experiments measured a singlet-excited state lifetime of 2.4 ns for the LOV2 domain.
Time-resolved fluorescence experiments utilizing a synchroscan streak camera revealed a singlet-excited state lifetime of the LOV2 domain of 2.4 ns.
singlet-excited state lifetime 2.4 ns
Fluorescence from the cryotrapped covalent adduct state of the LOV2 domain was extremely weak and had a spectrum maximum at 440 nm.
Fluorescence from the cryotrapped covalent adduct state was extremely weak, with a fluorescence spectrum showing a maximum at 440 nm.
fluorescence spectrum maximum 440 nm
The low-temperature absorption spectrum of the Avena sativa phototropin 1 LOV2 domain showed fine structure around 473 nm, indicating heterogeneity in the flavin binding pocket.
The low-temperature absorption spectrum of the LOV2 domain showed a fine structure around 473 nm, indicating heterogeneity in the flavin binding pocket.
absorption fine structure position 473 nm
Approval Evidence
6 sources18 linked approval claimsfirst-pass slug lov2-domain-of-avena-sativa-phototropin-1
Here, we study the influence of deletions and extensions of the A'α helix of the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) on the light-triggered structural response of the protein
Source:
the light-responsive LOV2 domain of Avena sativa phototropin 1
Source:
Using the photoresponse of the LOV2 domain of Avena sativa phototropin 1
Source:
Escherichia coli colonies expressing a mutagenized population of LOV2 derived from Avena sativa (oat) phot1 were screened for variants that showed altered photochemical reactivity in response to blue light excitation.
Source:
In the C-terminal LOV2 domain of Avena sativa phototropin 1
Source:
The spectroscopic properties of the LOV2 domain of phototropin 1 of Avena sativa (oat) have been investigated
Source:
functional rolesupports
The N-terminal and C-terminal helices of phototropin LOV2 domains are necessary for allosteric regulation of the phototropin kinase domain and may support signal integration of LOV1 and LOV2 domains.
It also suggests that the N- and C-terminal helices of phot-LOV2 domains are necessary for allosteric regulation of the phototropin kinase domain and may provide a basis for signal integration of LOV1 and LOV2 domains in phototropins.
Source:
interpretationsupports
The conformational changes in full-length phototropin LOV domains may be smaller than previously assumed, and full unfolding of the Jα helix in AsLOV2 constructs with short A'α helices may be a truncation artifact.
These results are different from shorter constructs, indicating that the conformational changes in full-length phototropin LOV domains might not be as large as previously assumed, and that the well-characterized full unfolding of the Jα helix in AsLOV2 with short A'α helices may be considered a truncation artifact.
Source:
mechanismsupports
In phototropin LOV2 domains, blue light illumination leads to covalent bond formation between protein and flavin that induces dissociation and unfolding of the C-terminal Jα helix and the N-terminal A'α helix.
In the second LOV domain of phototropins, called LOV2 domains, blue light illumination leads to covalent bond formation between protein and flavin that induces the dissociation and unfolding of a C-terminally attached α helix (Jα) and the N-terminal helix (A'α).
Source:
structure functionsupports
Deletion of the A'α helix abolishes light-induced unfolding of the Jα helix in AsLOV2.
Deletion of the A'α helix abolishes the light-induced unfolding of Jα
Source:
structure functionsupports
Extensions of the A'α helix attenuate the light-induced structural change of the Jα helix in AsLOV2.
whereas extensions of the A'α helix lead to an attenuated structural change of Jα
Source:
engineering strategysupports
LANS and LINX were engineered by embedding nuclear import or export sequence motifs into the C-terminal helix of the LOV2 domain of Avena sativa phototropin 1 to enable light-dependent trafficking of a target protein into and out of the nucleus.
We previously engineered two optogenetic systems, the light activated nuclear shuttle (LANS) and the light-inducible nuclear exporter (LINX), by embedding nuclear import or export sequence motifs into the C-terminal helix of the light-responsive LOV2 domain of Avena sativa phototropin 1, thus enabling light-dependent trafficking of a target protein into and out of the nucleus.
Source:
performance improvementsupports
Hinge-loop mutations in LOV2 were identified that favorably shift the dynamic range of the LANS and LINX photoswitches.
Here, we identify hinge-loop mutations that favorably shift the dynamic range (the ratio of the on- to off-target subcellular accumulation) of the LANS and LINX photoswitches.
Source:
engineering resultsupports
The authors developed LOV2-based analogues of kinase inhibitors whose activity is light dependent.
Using the photoresponse of the LOV2 domain of Avena sativa phototropin 1, we developed analogues of kinase inhibitors whose activity is light dependent.
Source:
mechanistic functionsupports
The LOV domain forms a flavin-cysteinyl adduct upon irradiation and thermally relaxes back to the ground state in the dark, functioning as a molecular switch controlling receptor kinase activity.
Irradiation of the LOV domain results in the formation of a flavin-cysteinyl adduct (LOV390) which thermally relaxes back to the ground state in the dark, effectively completing a photocycle that serves as a molecular switch to control receptor kinase activity.
Source:
structure function relationshipsupports
Decreasing the size of the isoleucine side chain accelerates adduct decay, indicating that steric interactions of this side chain stabilize the C-S cysteinyl adduct.
A kinetic acceleration trend was observed for adduct decay by decreasing the size of the isoleucine side chain. Our findings therefore indicate that the steric nature of this amino acid side chain contributes to stabilization of the C-S cysteinyl adduct.
Source:
variant effectsupports
A single Ile-to-Val substitution in the LOV2 domain caused slower LOV390 formation and adduct decay about one order of magnitude faster than wild type.
One variant showed slower rates of LOV390 formation but exhibited adduct decay times 1 order of magnitude faster than wild type. A single Ile --> Val substitution was responsible for the effects observed
Source:
comparative spectroscopic observationsupports
No spectral shifting of flavin emission was observed in the LOV2 domain or in FMN in aqueous solution.
No spectral shifting of the flavin emission was observed in the LOV2 domain nor in FMN in aqueous solution.
Source:
mechanistic propertysupports
Phototropins base their light-dependent action on the reversible formation of a covalent bond between an FMN cofactor and a conserved cysteine residue in LOV domains.
The phototropins are plant blue-light receptors that base their light-dependent action on the reversible formation of a covalent bond between a flavin mononucleotide (FMN) cofactor and a conserved cysteine residue in light, oxygen or voltage (LOV) domains.
Source:
spectroscopic measurementsupports
A pronounced phosphorescence emission around 600 nm was observed in the LOV2 domain between 77 and 120 K, placing the flavin triplet state at 16900 cm^-1.
A pronounced phosphorescence emission around 600 nm was observed in the LOV2 domain between 77 and 120 K, allowing for an accurate positioning of the flavin triplet state in the LOV2 domain at 16900 cm-1.
Source:
spectroscopic measurementsupports
The fluorescence quantum yield of the flavin cofactor in the LOV2 domain increased from 0.13 at room temperature to 0.41 at 77 K.
The fluorescence quantum yield of the flavin cofactor increased from 0.13 to 0.41 upon cooling the sample from room temperature to 77 K.
Source:
spectroscopic measurementsupports
Time-resolved fluorescence experiments measured a singlet-excited state lifetime of 2.4 ns for the LOV2 domain.
Time-resolved fluorescence experiments utilizing a synchroscan streak camera revealed a singlet-excited state lifetime of the LOV2 domain of 2.4 ns.
Source:
spectroscopic observationsupports
Fluorescence from the cryotrapped covalent adduct state of the LOV2 domain was extremely weak and had a spectrum maximum at 440 nm.
Fluorescence from the cryotrapped covalent adduct state was extremely weak, with a fluorescence spectrum showing a maximum at 440 nm.
Source:
spectroscopic observationsupports
The low-temperature absorption spectrum of the Avena sativa phototropin 1 LOV2 domain showed fine structure around 473 nm, indicating heterogeneity in the flavin binding pocket.
The low-temperature absorption spectrum of the LOV2 domain showed a fine structure around 473 nm, indicating heterogeneity in the flavin binding pocket.
Source:
Comparisons
Source-backed strengths
The domain has a well-characterized reversible photocycle based on irradiation-induced flavin-cysteinyl adduct formation followed by thermal dark-state relaxation. Its modularity is supported by successful domain-fusion designs such as LANS and LINX, and hinge-loop mutations were identified that improved the dynamic range of these trafficking photoswitches. Spectroscopic characterization further defines measurable photophysical properties, including a 2.4 ns singlet-excited-state lifetime and low-temperature flavin spectral features.
Source:
We previously engineered two optogenetic systems, the light activated nuclear shuttle (LANS) and the light-inducible nuclear exporter (LINX), by embedding nuclear import or export sequence motifs into the C-terminal helix of the light-responsive LOV2 domain of Avena sativa phototropin 1, thus enabling light-dependent trafficking of a target protein into and out of the nucleus.
Source:
Here, we identify hinge-loop mutations that favorably shift the dynamic range (the ratio of the on- to off-target subcellular accumulation) of the LANS and LINX photoswitches.
Source:
Using the photoresponse of the LOV2 domain of Avena sativa phototropin 1, we developed analogues of kinase inhibitors whose activity is light dependent.
Source:
No spectral shifting of the flavin emission was observed in the LOV2 domain nor in FMN in aqueous solution.
Ranked Citations
- 1.
- 2.
Derived from 3 linked claims. Example evidence: Irradiation of the LOV domain results in the formation of a flavin-cysteinyl adduct (LOV390) which thermally relaxes back to the ground state in the dark, effectively completing a photocycle that serves as a molecular switch to control receptor kinase activity.
- 3.
Derived from 2 linked claims. Example evidence: We previously engineered two optogenetic systems, the light activated nuclear shuttle (LANS) and the light-inducible nuclear exporter (LINX), by embedding nuclear import or export sequence motifs into the C-terminal helix of the light-responsive LOV2 domain of Avena sativa phototropin 1, thus enabling light-dependent trafficking of a target protein into and out of the nucleus.
- 4.