High-resolution structure of a naturally red-shifted LOV domain

Goncharov, Ivan M.; Smolentseva, Anastasia; Semenov, Oleg; Natarov, Ilia; Nazarenko, Vera V.; Yudenko, Anna; Remeeva, Alina; Gushchin, Ivan

doi:10.1016/j.bbrc.2021.06.046

Cited by 11 publications

(15 citation statements)

References 33 publications

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“…In pursuit of optogenetic control in deep biological tissue, several strategies aim at using red or NIR light to excite photoreceptors. Although residue exchanges around the chromophore could in principle induce a red-shift of absorbance spectra, such efforts had only modest success in flavin-based photoreceptors, arguably owing to the rigid scaffold of the isoalloxazine heterocycle (Goncharov et al, 2021;Röllen et al, 2021). By contrast, the color diversity realized across CBCRs and certain algal phytochromes (Rockwell et al, 2014;Fushimi and Narikawa, 2019) suggests that the chromophore and its absorbance spectrum are more malleable in the phytochrome superfamily.…”

Section: Perspectivesmentioning

confidence: 99%

Light-regulated gene expression in Bacteria: Fundamentals, advances, and perspectives

Ohlendorf

Möglich²

2022

Front. Bioeng. Biotechnol.

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Numerous photoreceptors and genetic circuits emerged over the past two decades and now enable the light-dependent i.e., optogenetic, regulation of gene expression in bacteria. Prompted by light cues in the near-ultraviolet to near-infrared region of the electromagnetic spectrum, gene expression can be up- or downregulated stringently, reversibly, non-invasively, and with precision in space and time. Here, we survey the underlying principles, available options, and prominent examples of optogenetically regulated gene expression in bacteria. While transcription initiation and elongation remain most important for optogenetic intervention, other processes e.g., translation and downstream events, were also rendered light-dependent. The optogenetic control of bacterial expression predominantly employs but three fundamental strategies: light-sensitive two-component systems, oligomerization reactions, and second-messenger signaling. Certain optogenetic circuits moved beyond the proof-of-principle and stood the test of practice. They enable unprecedented applications in three major areas. First, light-dependent expression underpins novel concepts and strategies for enhanced yields in microbial production processes. Second, light-responsive bacteria can be optogenetically stimulated while residing within the bodies of animals, thus prompting the secretion of compounds that grant health benefits to the animal host. Third, optogenetics allows the generation of precisely structured, novel biomaterials. These applications jointly testify to the maturity of the optogenetic approach and serve as blueprints bound to inspire and template innovative use cases of light-regulated gene expression in bacteria. Researchers pursuing these lines can choose from an ever-growing, versatile, and efficient toolkit of optogenetic circuits.

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Section: Perspectivesmentioning

confidence: 99%

Light-regulated gene expression in Bacteria: Fundamentals, advances, and perspectives

Ohlendorf

Möglich²

2022

Front. Bioeng. Biotechnol.

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“…Several studies recently also provided a molecular characterization of the spectral effects of mutations on iLOV to gain a mechanistic understanding. − Röllen and colleagues have shown that iLOV V392T/Q489K fluorescence has a slight red-shift with an emission spectrum maximum of 502 nm . Similar mutations on iLOV homolog protein CagFbFP I52T/Q148K with the emission maximum of 504 nm and another blue-shifted variant CagFbFP Q148K with the emission maximum of 491 nm were used in fluorescence microscopy experiments and were successfully spectrally separated .…”

Section: Discussionmentioning

confidence: 99%

Experimental Characterization of In Silico Red-Shift-Predicted iLOV^L470T/Q489K and iLOV^{V392K/F410V/A426S} Mutants

et al. 2022

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iLOV is a flavin mononucleotide-binding fluorescent protein used for in vivo cellular imaging similar to the green fluorescent protein. To expand the range of applications of iLOV, spectrally tuned red-shifted variants are desirable to reduce phototoxicity and allow for better tissue penetration. In this report, we experimentally tested two iLOV mutants, iLOV L470T/Q489K and iLOV V392K/F410V/A426S , which were previously computationally proposed by ( Khrenova Khrenova 28992704 J. Phys. Chem. B 2017 121 10018 10025 ) to have red-shifted excitation and emission spectra. While iLOV L470T/Q489K is about 20% brighter compared to the WT in vitro , it exhibits a blue shift in contrast to quantum mechanics/molecular mechanics (QM/MM) predictions. Additional optical characterization of an iLOV V392K mutant revealed that V392 is essential for cofactor binding and, accordingly, variants with V392K mutation are unable to bind to FMN. iLOV L470T/Q489K and iLOV V392K/F410V/A426S are expressed at low levels and have no detectable fluorescence in living cells, preventing their utilization in imaging applications.

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“…Previously, several attempts have been made to generate flavin-based fluorescent proteins with shifted spectra, with moderate success (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). In this work, we mutated a previously untargeted amino acid position, that of photoactive cysteine (corresponding to A85 in CagFbFP), in addition to mutations of other residues near the flavin (I52 and Q148, Figure 3A), and identified a palette of 22 variants that uniformly cover the range of emission spectra with the maxima from 486 to 512 nm (Figure 3C).…”

Section: Discussionmentioning

confidence: 99%

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging

Николаев

Yudenko

Smolentseva

et al. 2022

Preprint

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Flavin-binding fluorescent proteins (FbFPs) are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of twenty-two finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans (CagFbFP). We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of FbFPs in fluorescence microscopy.

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High-resolution structure of a naturally red-shifted LOV domain

Cited by 11 publications

References 33 publications

Light-regulated gene expression in Bacteria: Fundamentals, advances, and perspectives

Light-regulated gene expression in Bacteria: Fundamentals, advances, and perspectives

Experimental Characterization of In Silico Red-Shift-Predicted iLOV^L470T/Q489K and iLOV^{V392K/F410V/A426S} Mutants

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging

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High-resolution structure of a naturally red-shifted LOV domain

Cited by 11 publications

References 33 publications

Light-regulated gene expression in Bacteria: Fundamentals, advances, and perspectives

Light-regulated gene expression in Bacteria: Fundamentals, advances, and perspectives

Experimental Characterization of In Silico Red-Shift-Predicted iLOVL470T/Q489K and iLOVV392K/F410V/A426S Mutants

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging

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Product

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Experimental Characterization of In Silico Red-Shift-Predicted iLOV^L470T/Q489K and iLOV^{V392K/F410V/A426S} Mutants