Light-activated proteins

Curley, Kieran; Lawrence, David S.

doi:10.1016/s1367-5931(99)80015-5

Cited by 72 publications

(43 citation statements)

References 17 publications

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“…Thus far, reports about caged proteins that were used in biological systems have been limited (25,32). For example, NVOC-Cl was used to reversibly block polymerization activity of G-actin (24), to control the transcriptional activity of GAL4VP16 in living Drosophila embryos (33), or to study the kinetics of intracellular signaling pathways by using caged protein kinases or their inhibitors (34).…”

Section: Discussionmentioning

confidence: 99%

A caged Ab reveals an immediate/instructive effect of BDNF during hippocampal synaptic potentiation

Kossel

Cambridge

Wagner

et al. 2001

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

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Neurotrophins have been shown to be involved in functional strengthening of central nervous system synapses. Although their general importance in this process is undisputed, it remains unresolved whether neurotrophins are truly mediators of synaptic strengthening or merely important cofactors. To address this question, we have devised a method to inactivate endogenous brain-derived neurotrophic factor (BDNF) with high time resolution by “caging” a function-blocking mAb against BDNF with a photosensitive protecting compound. Different assays were used to show that this inactivation of the Ab is reversible by UV light. Synaptic potentiation after τ-burst stimulation in the CA1 region of acute hippocampal slices was significantly less when applying the unmodified Ab compared with the caged Ab. Importantly, photoactivation of the caged Ab during the time of induction of synaptic enhancement led to a marked decrease in potentiation. Our experiments therefore strengthen the view that endogenous BDNF has fast effects during induction of synaptic plasticity. The results additionally show that caged Abs can provide a tool for precise spatiotemporal control over endogenous protein levels.

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Section: Discussionmentioning

confidence: 99%

A caged Ab reveals an immediate/instructive effect of BDNF during hippocampal synaptic potentiation

Kossel

Cambridge

Wagner

et al. 2001

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

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“…The installation of light-removable protecting groups on biological macromolecules is a versatile approach to the photochemical regulation of RNA, DNA, and protein activity. [4][5][6][7][8][9][10] For the generation of caged proteins in live cells, the genetic code can be expanded allowing for the site-specific incorporation of caged amino acids into proteins. [11][12][13][14][15][16][17][18][19][20] The caged amino acids are inserted in response to an amber stop codon, TAG, by suppressor tRNAs that have been misacylated by engineered tRNA synthetases.…”

Section: Introductionmentioning

confidence: 99%

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

et al. 2013

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Photocaging provides a method to spatially and temporally control biological function and gene expression with high resolution. Proteins can be photochemically controlled through the sitespecific installation of caging groups on amino acid side chains that are essential for protein function. The photocaging of a synthetic gene network using unnatural amino acid mutagenesis in mammalian cells was demonstrated with an engineered bacteriophage RNA polymerase. A caged T7 RNA polymerase was expressed in cells with an expanded genetic code and used in the photochemical activation of genes under control of an orthogonal T7 promoter, demonstrating tight spatial and temporal control. The synthetic gene expression system was validated with two reporter genes (luciferase and EGFP) and applied to the light-triggered transcription of short hairpin RNA constructs for the induction of RNA interference.

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“…[8] For time-resolved studies it is necessary to trigger the enzymatic activity with precise time control, which can be achieved by a photochemical release of caged substrates or enzymes. [9] Besides other technical difficulties inherent to Laue crystallography, this method requires an efficient and synchronous photofragmentation reaction throughout a protein crystal. To overcome these technical difficulties we developed an alternative cryophotolytic [ method.…”

Section: Introductionmentioning

confidence: 99%

Photoreversible Inhibition of Cholinesterases: Catalytic Serine‐Labeled Caged Butyrylcholinesterase

et al. 2003

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The photoregulation of the catalytic activity of butyrylcholinesterase (BChE) was investigated by treating the enzyme with a newly developed carbamylating reagent, N-methyl-N-(2-nitrophenyl)carbamoyl chloride, which has proved to be an efficient photoremovable alcohol-protecting group. BChE was efficiently inhibited in a time- and concentration-dependent manner, and the enzyme could be protected against inhibition by active-site-specific ligands (that is, tacrine). The inactivation kinetics showed a biphasic character, which can be analyzed as the result of the existence of two conformational states in solution. Pseudo-irreversible inactivation of BChE, which results from catalytic serine carbamylation, was suggested by recovery of the enzyme activity after dilution and was demonstrated by X-ray crystallography. Remarkably, the 3D structure of the carbamylated BChE conjugate showed a nonambiguous carbamylation of the catalytic serine residue as the only chemical modification on the protein. The photoreversibility of the enzyme inactivation was analyzed by irradiating the inactivated enzyme at 365 nm and was shown to reach completion in some conditions. The efficient and specific "caging" of BChE, together with the availability of carbamylated BChE crystals, will offer a unique possibility to study the catalytic properties of this enzyme by kinetic crystallography after cryophotolytic uncaging of the enzyme conjugate crystals.

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Light-activated proteins

Cited by 72 publications

References 17 publications

A caged Ab reveals an immediate/instructive effect of BDNF during hippocampal synaptic potentiation

A caged Ab reveals an immediate/instructive effect of BDNF during hippocampal synaptic potentiation

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

Photoreversible Inhibition of Cholinesterases: Catalytic Serine‐Labeled Caged Butyrylcholinesterase

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