| 1. |
- Kipper, Kalle, et al.
(författare)
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Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI
- 2018
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Ingår i: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 13:6
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Tidskriftsartikel (refereegranskat)abstract
- The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis- based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O-1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.
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| 2. |
- Marter, Kathrin, et al.
(författare)
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Inhibition of Protein Synthesis with Highly Soluble Caged Compounds
- 2017
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Ingår i: ChemistrySelect. - : Wiley. - 2365-6549. ; 2:22, s. 6212-6217
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Tidskriftsartikel (refereegranskat)abstract
- To target protein synthesis in defined areas, e.g. neuropiles of small brains or subcellular structures, locally restricted inhibition of protein synthesis is needed and can be realized by caged compounds of protein synthesis inhibitors (PSI). Since organic solvents interfere with protein synthesis themselves, the use of water-soluble caged PSIs is a prerequisite in studies on protein synthesis. Such compounds are sparsely available. We developed and characterized efficient highly soluble caged compounds of the PSIs anisomycin and emetine masking their biological activity with a {8-[bis(carboxymethyl)aminomethyl]-6-bromo-7-hydroxycoumarin-4-yl}methoxycarbonyl (BBHCMOC) derivative. The absorption spectra of the resulting BBHCMOC-caged anisomycin and BBHCMOC-caged emetine show long-wavelength maxima and the extinction coefficients are high, allowing uncaging under non-damaging light conditions. When uncaged, these caged PSIs reliably inhibit protein synthesis in an in vitro translation system and in cell culture. Taken the whole spectrum of properties into account, our BBHCMOC-caged PSIs are highly qualified for in vivo studies.
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| 3. |
- Schaal, J., et al.
(författare)
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Coumarin-based octopamine phototriggers and their effects on an insect octopamine receptor
- 2012
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 13:10, s. 1458-1464
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Tidskriftsartikel (refereegranskat)abstract
- We have developed and characterized efficient caged compounds of the neurotransmitter octopamine. For derivatization, we introduced [6-bromo-8-(diethylaminomethyl)-7-hydroxycoumarin-4-yl]methoxycarbonyl (DBHCMOC) and {6-bromo-7-hydroxy-8-[(piperazin-1-yl)methyl]coumarin-4-yl}methoxycarbonyl (PBHCMOC) moieties as novel photo-removable protecting groups. The caged compounds were functionally inactive when applied to heterologously expressed octopamine receptors (AmOctα1R). Upon irradiation with UV–visible or IR light, bioactive octopamine was released and evoked Ca2+ signals in AmOctα1R-expressing cells. The pronounced water solubility of compounds 2–4 in particular holds great promise for these substances as excellent phototriggers of this important neurotransmitter.
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