Henlea earthworm bioluminescence comprises violet-blue BRET from tryptophan 2-carboxylate to deazaflavin cofactor.

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).

Conjugated Dienoic Acid Peroxides as Substrates in Chaetopterus Bioluminescence System.

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 Fe2+ 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.

Far-red fluorescent tags for protein imaging in living tissues.

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.

Bright monomeric red fluorescent protein with an extended fluorescence lifetime.

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.