Localization in vivo with fluorescent amino acids
The fluorescent amino acid L-(7-hydroxycoumarin-4-yl) (CouAA) ethylglycine is relatively small, has a high fluorescence quantum yield and relatively large Stoke's shift. It is also solvent polar and pH-sensitive so it can indicate pH-changes in the cell (Wang et al., 2006). The amino acid is suitable for in vivo and in vitro localization, and in contrary to fluorescent proteins even for localization in SDS PAGES. For a detailed description of our labeling tool please refer to the labeling page of the toolkit.
Analyzing of intermolecular distances in proteins with non-canonical amino acids
The first step is the incorporation of the non-canonical amino acids. In proteins naturally containing no cysteins (cysteines are the only canonical amino acids that could be labeled specific) or in which the exchanges of cysteines do not influence the structure only one ncAA and one cysteine at specific points need to be incorporated to be labeled. In proteins that contain cysteine, two ncAAs need to be incorporated for the labeling (Kim et al., 2013).
Non-canonical amino acids could be incorporated by orthogonal tRNA/aaRS synthetases in response to the amber stop codon. However, this allows only the incorporation of one noncanonical amino acid. To incorporate the second amino acid, another orthogonal amino acid could be used for the incorporation in response to a rarely used leucine codon. For structural analysis the amino acids are specific labeled with chromophores. This labeling is possible due to the functional groups of the amino acids which could form a covalent bond to the fluorophores in a chemical reaction. After the protein is labeled, the fluorescence of the chromophores could be measured to draw conclusions on the distance of the ncAA from each other (Brustad et al., 2008, Kim et al., 2013).
To incorporate the ncAAs, we provide three different tRNA/aminoacyl-synthetases which incorporate in response to the amber or the less used leucine codon. For more details please refer to the labeling page.
Immobilization and fusing of proteins with non-canonical amino acids
Protein regulation with photoswitching and photolysis amino acids
Photocaging: a “protection” group facilitates or inhibits the normal function of a given protein, but after cleavage of the chemical moiety the protein of interest is de/activated. This process is irreversible.
Photoswitching: the chemical moiety used can be switched between “ON/OFF” stages. This process is reversible. For both approaches the incorporation of a chemical moiety into a permissive site of the protein of interest is accomplished through amber suppressor tRNA (Bose et al., 2006).
The advantage of light as the trigger for the cleaving and conformational change lies in its highly controllable, selective and inexpensive application. In contrast to chemical substrates used for induction of a reaction, light does not leave residues which themselves can influence the test environment. Furthermore, many already established techniques can be adapted to apply the specific wavelength and irradiation time for any possible non-canonical amino acid (Brieke et al., 2012).
We decided to use a photoisoerisable amino acid because of the reversible reaction. To demonstrate this tool we incorporated the photoisomerisable amino acid p-azobenzene in the enzyme CrtI to regulate the lycopene pathway. We showed that the activity of CrtI could be regulated only by light irradiation.
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Charbon, G., Brustad, E., Scott, K.A., Wang, J., Lobner-Oelson, A. Schultz, P. G., Jacobs-Wagner, C., Chapman, E.(2011). Subcellular Protein Localization by Using a Genetically Encoded Fluorescent Amino Acid. ChemBioChem. 12:1818-1821.
Charbon, G., Wang, J., Brustad, E., Schultz, P. G., Horwiich, A. L., Jacobs-Wagner, C., Chapman, E.(2011). Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid. Bioorg Med Chem Lett. 21(20): 6067-6070.
Kim, J., Seo, M., Lee, S., Cho, K., Yang, A., Woo, K., Kim, H., Park, H.(2012). Simple and Efficient Strategy for Site-Specific Dual Labeling of Proteins for Single-Molecule Fluorescence Resonance Energy Transfer Analysis. Analytical Chemistry.85: 1468-1474.
Klán, P., Šolomek, T., Bochet, C.G., Blanc, A., Givens, R., Rubina, M., Popik, V., Kostikov, A., and Wirz, J. (2013). Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy. Chem. Rev. 113: 119–191.
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