A vast colour palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility and interactions. However, low brightness has remained a problem in far-red ...variants, which hampers multicolour labelling and whole-body imaging techniques. In the present paper, we report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high-brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. We also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-IR fluorescence compared with mRaspberry or mCherry, and is 20-fold brighter than mPlum. Together, monomeric mKate2 and pseudo-monomeric tdKatushka2 represent the next generation of extra-bright far-red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals.
We recently identified the deazaflavin cofactor as a light emitter in novel bioluminescence (BL) system from Siberian earthworms Henlea sp. (Petushkov et al., 2023, Org. Biomol. Chem. 21:415–427). In ...the present communication we compared in vitro BL spectra in the absence and in the presence of the cofactor and found a wavelength shift from 420 to 476 nm. This violet-blue BRET to deazaflavin cofactor (acceptor of photonless transfer) masks the actual oxyluciferin as an emitter (BRET donor) in the novel BL system. The best candidate for that masked chromophore is tryptophan 2-carboxylate (T2C) found previously as a building block in some natural products isolated from Henlea sp. (Dubinnyi et al., 2020, ChemSelect 5:13155–13159). We synthesized T2C and acetyl-T2C, verified their presence in earthworms by nanoflow-HRMS, explored spectral properties of excitation and emission spectra and found a chain of excitation/emission maxima with a perfect potential for BRET: 300 nm (excitation of T2C) - 420 nm (emission of T2C) - 420 nm (excitation of deazaflavin) - 476 nm (emission of deazaflavin, BL). An array of natural products with T2C chromophore are present in BL earthworms as candidates for novel oxyluciferin. We demonstrated for the Henlea BL that the energy of the excited state of the T2C chromophore is transferred by the Förster mechanism and then emitted by deazaflavin (BRET), similarly to known examples: aequorin-GFP in Aequorea victoria and antenna proteins in bacterial BL systems (lumazine from Photobacterium and yellow fluorescent protein from Vibrio fischeri strain Y1).
•Tryptophan 2-carboxylates are widespread compounds in bioluminescent Henlea earthworms.•The tryptophan 2-carboxylate was synthesized and unambiguously characterized by NMR, MS and UV for the first time.•The hypothesis of tryptophan 2-carboxylate-to-deazaflavin FRET is formulated for novel bioluminescent system.•Bioluminescence spectra with and without deazaflavin were measured and confirmed the violet-blue BRET in Henlea.•The BRET in nature was previously reported only as aequorin-GFP transfer and for antenna proteins in bacterial BL systems.
Fluorescent proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of proteins in living cells. Here we report TagRFP, ...a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.
Biochemistry of bioluminescence of the marine parchment tubeworm
has been in research focus for over a century; however, the results obtained by various groups contradict each other. Here, we report ...the isolation and structural elucidation of three compounds from
algae, which demonstrate bioluminescence activity with
luciferase in the presence of Fe
ions. These compounds are derivatives of polyunsaturated fatty acid peroxides. We have also obtained their structural analogues and demonstrated their activity in the bioluminescence reaction, thus confirming the broad substrate specificity of the luciferase.
Biochemistry of bioluminescence of the marine parchment tubeworm Chaetopterus has been in research focus for over a century; however, the results obtained by various groups contradict each other. ...Here, we report the isolation and structural elucidation of three compounds from Chaetomorpha linum algae, which demonstrate bioluminescence activity with Chaetopterus luciferase in the presence of Fesup.2+ ions. These compounds are derivatives of polyunsaturated fatty acid peroxides. We have also obtained their structural analogues and demonstrated their activity in the bioluminescence reaction, thus confirming the broad substrate specificity of the luciferase.
The Fe–C system, which is widely used to grow commercial high‐pressure–high‐temperature diamond monocrystals, is rather complicated due to the formation of carbides. The carbide Fe3C is a normal run ...product, but the pressure at which Fe7C3 carbide becomes stable is a subject of discussion. This paper demonstrates the synthesis of Fe7C3 carbide and its detailed study using single‐crystal and powder X‐ray diffraction, as well as electron probe micro‐analysis and scanning electron microscopy. The experiments were performed using a multiple‐anvil high‐pressure apparatus of `split‐sphere' (BARS) type at a pressure of 5.5 GPa and a temperature of 1623 K. Our results show that in the Fe–C system, in addition to diamond, a phase that corresponds to the Fe7C3 carbide was synthesized. This means that both carbides (Fe7C3 and Fe3C) are stable at 5.5 GPa. Two crystal phases are described, Fe14C6 and Fe28C12−x. Fe14C6 is based on the well known rhombic structure of Fe7C3, while Fe28C12−x has a different packing order of Fe6C polyhedrons. The results obtained in this study should be taken into account when synthesizing and growing diamond at high pressures and temperatures in metal–carbon systems with a high iron content, as well as when conducting experimental studies on the synthesis of diamond directly from carbide.
Experiments on diamond synthesis in the Fe–C system at 5.5 GPa and 1623 K were performed. X‐ray diffraction analysis showed the formation of two carbides, Fe3C and Fe7C3. Two polytypic modifications of Fe7C3 are described, namely Fe14C6 and Fe28C12−x.
The Fe–C system, which is widely used to grow commercial high-pressure–high-temperature diamond monocrystals, is rather complicated due to the formation of carbides. The carbide Fe
3
C is a normal ...run product, but the pressure at which Fe
7
C
3
carbide becomes stable is a subject of discussion. This paper demonstrates the synthesis of Fe
7
C
3
carbide and its detailed study using single-crystal and powder X-ray diffraction, as well as electron probe micro-analysis and scanning electron microscopy. The experiments were performed using a multiple-anvil high-pressure apparatus of `split-sphere' (BARS) type at a pressure of 5.5 GPa and a temperature of 1623 K. Our results show that in the Fe–C system, in addition to diamond, a phase that corresponds to the Fe
7
C
3
carbide was synthesized. This means that both carbides (Fe
7
C
3
and Fe
3
C) are stable at 5.5 GPa. Two crystal phases are described, Fe
14
C
6
and Fe
28
C
12−
x
. Fe
14
C
6
is based on the well known rhombic structure of Fe
7
C
3
, while Fe
28
C
12−
x
has a different packing order of Fe
6
C polyhedrons. The results obtained in this study should be taken into account when synthesizing and growing diamond at high pressures and temperatures in metal–carbon systems with a high iron content, as well as when conducting experimental studies on the synthesis of diamond directly from carbide.
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