The Incorporation of a Photoisomerizable Amino Acid into Proteins in E. coli

Bose, Mohua; Groff, Dan; Xie, Jianming; Brustad, Eric M.; Schultz, Peter G.

doi:10.1021/ja055467u

Cited by 162 publications

(157 citation statements)

References 21 publications

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“…Examples of biomolecular systems into which azobenzene has been incorporated for photo-regulation are peptides [1][2][3] , enzymes [4][5][6][7] , oligonucleotides 8,9 and ion channels 10,11 . Methods for incorporating azobenzene chromophores into biomolecules include solid-phase peptide or oligonucleotide synthesis 7,12 , nonsense suppression via azobenzenecharged suppressor tRNAs 4 , and both non-selective 13 and targeted chemical modification of protein side chains 14 .…”

Section: Introductionmentioning

confidence: 99%

Synthesis of 3,3′-bis(sulfonato)-4,4′-bis(chloroacetamido)azobenzene and cysteine cross-linking for photo-control of protein conformation and activity

2007

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Section: Introductionmentioning

confidence: 99%

Synthesis of 3,3′-bis(sulfonato)-4,4′-bis(chloroacetamido)azobenzene and cysteine cross-linking for photo-control of protein conformation and activity

2007

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“…Azobenzene can be reversibly isomerized between the extended trans-and the more compact cis-configuration by illumination with UV (trans → cis) or blue-light (cis → trans) as well as by thermal relaxation (cis → trans) (2-4). Four generally applicable approaches have been used to introduce azobenzene groups into peptides or proteins: (i) incorporation during peptide synthesis (5)(6)(7)(8), (ii) incorporation during in vitro translation (9,10), (iii) incorporation in vivo by using an orthogonal tRNA/aminoacyl tRNA synthetase pair specific for phenylalanine-4′-azobenzene (11), and (iv) chemical modification of peptides and proteins (3,12,13). Another more specific approach is to use azobenzenemodified ligands (e.g., inhibitors) for proteins (14,15).…”

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confidence: 99%

Controlling the enzymatic activity of a restriction enzyme by light

Schierling

Noël

Wende

et al. 2009

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

165

164

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For many applications it would be desirable to be able to control the activity of proteins by using an external signal. In the present study, we have explored the possibility of modulating the activity of a restriction enzyme with light. By cross-linking two suitably located cysteine residues with a bifunctional azobenzene derivative, which can adopt a cis-or trans-configuration when illuminated by UV or blue light, respectively, enzymatic activity can be controlled in a reversible manner. To determine which residues when crosslinked show the largest "photoswitch effect," i.e., difference in activity when illuminated with UV vs. blue light, >30 variants of a single-chain version of the restriction endonuclease PvuII were produced, modified with azobenzene, and tested for DNA cleavage activity. In general, introducing single cross-links in the enzyme leads to only small effects, whereas with multiple cross-links and additional mutations larger effects are observed. Some of the modified variants, which carry the cross-links close to the catalytic center, can be modulated in their DNA cleavage activity by a factor of up to 16 by illumination with UV (azobenzene in cis) and blue light (azobenzene in trans), respectively. The change in activity is achieved in seconds, is fully reversible, and, in the case analyzed, is due to a change in V max rather than K m .azobenzene | DNA cleavage | endonuclease | photoswitch | PvuII P roteins exist in nature whose activity can be controlled by light; perhaps one of the best known examples is rhodopsin, which is regulated by the cis∕trans isomerization of its cofactor retinal. For many biological applications it would be desirable to selectively switch the activity of a protein on and off by light in a similar manner (1). This could be accomplished by the introduction of a photosensitive compound into the protein of interest. Recent developments in photosensitive compounds such as the azobenzene derivatives have made the scenario a reality. Azobenzene can be reversibly isomerized between the extended trans-and the more compact cis-configuration by illumination with UV (trans → cis) or blue-light (cis → trans) as well as by thermal relaxation (cis → trans) (2-4). Four generally applicable approaches have been used to introduce azobenzene groups into peptides or proteins: (i) incorporation during peptide synthesis (5-8), (ii) incorporation during in vitro translation (9, 10), (iii) incorporation in vivo by using an orthogonal tRNA/aminoacyl tRNA synthetase pair specific for phenylalanine-4′-azobenzene (11), and (iv) chemical modification of peptides and proteins (3,12,13). Another more specific approach is to use azobenzenemodified ligands (e.g., inhibitors) for proteins (14,15). Chemical modification, the most widely used of these approaches, can be done with mono-or bifunctional azobenzene derivatives. Modification with monofunctional azobenzene derivatives relies on steric effects (e.g., interference with ligand binding), whereas modification with bifunctional azobenzene derivat...

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“…In this case, reversible photochemically induced changes in the shape or electronic character of functionally important amino acids have been used to control the function of proteins in response to light (8)(9)(10) or to alter the backbone structure of peptides (11), thereby controlling their interaction with other biological macromolecules (12). In an alternative strategy, photoisomerization of a tethered ligand can be used to reversibly present, and withdraw, a ligand from a binding site.…”

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confidence: 99%

Mechanisms of photoswitch conjugation and light activation of an ionotropic glutamate receptor

Gorostiza

Volgraf²,

Numano

et al. 2007

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

164

228

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The analysis of cell signaling requires the rapid and selective manipulation of protein function. We have synthesized photoswitches that covalently modify target proteins and reversibly present and withdraw a ligand from its binding site due to photoisomerization of an azobenzene linker. We describe here the properties of a glutamate photoswitch that controls an ion channel in cells. Affinity labeling and geometric constraints ensure that the photoswitch controls only the targeted channel, and enables spatial patterns of light to favor labeling in one location over another. Photoswitching to the activating state places a tethered glutamate at a high (millimolar) effective local concentration near the binding site. The fraction of active channels can be set in an analog manner by altering the photostationary state with different wavelengths. The bistable photoswitch can be turned on with millisecond-long pulses at one wavelength, remain on in the dark for minutes, and turned off with millisecond long pulses at the other wavelength, yielding sustained activation with minimal irradiation. The system provides rapid, reversible remote control of protein function that is selective without orthogonal chemistry.azobenzene ͉ ion channel ͉ photoisomerization ͉ optical switch ͉ remote control M uch progress has been made recently in the real-time noninvasive detection of protein function (1), but the development of approaches for remote protein manipulation within the complex environment of the cell has lagged behind. A major advance has been the development of photolysable cages for soluble ligands (2). The caged ligand is allowed to slowly diffuse into tissue in its inert form, and a powerful light flash cleaves a photolabile protecting group, releasing the active ligand rapidly (within microseconds) (3,4). This provides fast on-rates that are complemented with reasonably fast off-rates, which depend on native binding affinity, diffusion, sequestration, and breakdown. However, because native ligands often act on multiple proteins, this approach has limited selectivity. Introducing foreign receptors that bind nonnative ligands, on the other hand, enables cellular stimulation without the activation of endogenous proteins (5, 6) and can even be used in a photolysable form for rapid release (7).Photoisomerizable moieties have also found use in the remote and selective control of native protein function. In this case, reversible photochemically induced changes in the shape or electronic character of functionally important amino acids have been used to control the function of proteins in response to light (8-10) or to alter the backbone structure of peptides (11), thereby controlling their interaction with other biological macromolecules (12). In an alternative strategy, photoisomerization of a tethered ligand can be used to reversibly present, and withdraw, a ligand from a binding site. To date, several ion channel photoswitches have been reported wherein a ligand is tethered to the channel via a linker containing a photoisom...

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The Incorporation of a Photoisomerizable Amino Acid into Proteins in E. coli

Cited by 162 publications

References 21 publications

Synthesis of 3,3′-bis(sulfonato)-4,4′-bis(chloroacetamido)azobenzene and cysteine cross-linking for photo-control of protein conformation and activity

Synthesis of 3,3′-bis(sulfonato)-4,4′-bis(chloroacetamido)azobenzene and cysteine cross-linking for photo-control of protein conformation and activity

Controlling the enzymatic activity of a restriction enzyme by light

Mechanisms of photoswitch conjugation and light activation of an ionotropic glutamate receptor

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