Impact of binding to the multidrug resistance regulator protein LmrR on the photo-physics and -chemistry of photosensitizers

Mejías, Sara H.; Roelfes, Gérard; Browne, Wesley R.

doi:10.1039/d0cp01755h

Cited by 13 publications

(10 citation statements)

References 90 publications

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“…We envisioned that binding of planar organic fluorophores into the hydrophobic binding pockets of LmrR 33 and RamR (hereafter named CTPEs) would improve their photophysical properties. Furthermore, engineered variants of LmrR were used to minimize interactions of the tags with DNA (see Methods section).…”

Section: Resultsmentioning

confidence: 99%

Chemogenetic Tags with Probe Exchange for Live-Cell Fluorescence Microscopy

et al. 2021

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Fluorogenic protein tagging systems have been less developed for prokaryotes than for eukaryotic cell systems. Here, we extend the concept of noncovalent fluorogenic protein tags in bacteria by introducing transcription factor-based tags, namely, LmrR and RamR, for probe binding and fluorescence readout under aerobic and anaerobic conditions. We developed two chemogenetic protein tags that impart fluorogenicity and a longer fluorescence lifetime to reversibly bound organic fluorophores, hence the name Chemogenetic Tags with Probe Exchange (CTPEs). We present an extensive characterization of 30 fluorophores reversibly interacting with the two different CTPEs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of these tags. The reversible binding of organic fluorophores to the CTPEs and the superior photophysical properties of organic fluorophores enable long-term fluorescence microscopy of living bacterial cells. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein–protein interactions, and prolonged live-cell microscopy, even under oxygen-free conditions.

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Section: Resultsmentioning

confidence: 99%

Chemogenetic Tags with Probe Exchange for Live-Cell Fluorescence Microscopy

et al. 2021

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“…We envisioned that seclusion of planar organic fluorophores into the hydrophobic binding pockets within LmrR (Mejías et al, 2020) and RamR (hereafter named oxygen-independent chemogenetic protein (OICP) tags) could improve their photophysical properties, which depends on the distinct chemical environment of the protein binding sites. Furthermore, we Table 1 and Supplementary Figs.…”

Section: Resultsmentioning

confidence: 99%

Oxygen-independent chemogenetic protein tags for live-cell fluorescence microscopy

Iyer

Baranov

Foster

et al. 2020

Preprint

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Fluorescent proteins enable targeted visualization of biomolecules in living cells, but their maturation is oxygen-dependent and they are susceptible to aggregation and/or suffer from poor photophysical properties. Organic fluorophores are oxygen-independent with superior photophysical properties, but targeting biomolecules in vivo is challenging. Here, we introduce two oxygen-independent chemogenetic protein (OICP) tags that impart fluorogenicity and fluorescence lifetime enhancement to bound organic dyes. We present a photo- and physicochemical characterization of thirty fluorophores interacting with two OICPs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of the proteins. The binding specificity of the tags and the superior photophysical properties of organic fluorophores enable microscopy of living bacterial and eukaryotic cells. The exchange of photobleached dye for unbleached fluorophore enables prolonged live-cell imaging. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein-protein interactions, and microscopy applications under strictly oxygen-free conditions.

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“…The λ max values in the absorbance and emission spectra of the cofactors did not change significantly upon bioconjugation (Supplementary Figure 4-5), consistent with the fact that the orbital energies of Ru(Bpy) 3 2+ and related complexes are generally not sensitive to changes in solvent/environment 46 . The luminescence lifetimes of the ArMs were also evaluated since it is known that the lifetime of luminophores like Ru(Bpy) 3 2+ increases in more hydrophobic environments, 30,47,48 such as that expected within the POP active site, and substantial increases compared to the free complexes were observed (Table 1). Finally, CD spectroscopy was used to more directly interrogate possible interactions between 1a-f and the different protein scaffolds (Figure 2C and Supplementary Figure 6).…”

Section: Covalent Arm Preparation and Characterizationmentioning

confidence: 99%

Controlling the optical and catalytic properties of artificial metalloenzyme photocatalysts using chemogenetic engineering

Zubi

Liu

et al. 2022

Chem. Sci.

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Impact of binding to the multidrug resistance regulator protein LmrR on the photo-physics and -chemistry of photosensitizers

Abstract: Binding of photosensitizing compounds to the LmrR protein with and without tryptophan residues has a profound and varied effect on their photochemistry and ability to generate reactive oxygen species.

Cited by 13 publications

References 90 publications

Chemogenetic Tags with Probe Exchange for Live-Cell Fluorescence Microscopy

Chemogenetic Tags with Probe Exchange for Live-Cell Fluorescence Microscopy

Oxygen-independent chemogenetic protein tags for live-cell fluorescence microscopy

Controlling the optical and catalytic properties of artificial metalloenzyme photocatalysts using chemogenetic engineering

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