The deep learning technologies have become the driver of the revolutionary changes in scientific research in various fields. The AlphaFold-2 neural network software development that has solved the semicentennial problem of 3D protein structure prediction based on primary amino acid sequence is the most obvious example of using such technologies in structural biology and biomedicine. The use of deep learning methods for the prediction of protein–ligand interactions can considerably simplify predicting, speed up the development of new effective pharmaceuticals and change the concept of drug design.

Tekhnologii glubokogo obucheniya stali drajverom revolyucionnyh izmenenij v nauchnyh issledovaniyah raznyh oblastej. Naibolee yarkim primerom ih primeneniya v oblasti strukturnoj biologii i biomediciny yavlyaetsya programmnaya razrabotka nejroset' AlphaFold-2, reshivshaya poluvekovuyu problemu predskazaniya 3D-struktury belkov po pervichnoj aminokislotnoj posledovatel'nosti. Ispol'zovanie metodov glubokogo obucheniya dlya predskazaniya belok-ligandnyh vzaimodejstvij smozhet znachitel'no uprostit' predskazanie, uskorit' razrabotku novyh effektivnyh lekarstvennyh preparatov i pomenyat' koncepciyu drag-dizajna.

The recently described bioluminescent system from fungi has great potential for developing highly efficient tools for biomedical research. Luciferase enzyme is one of the most crucial components of this system. The luciferase from Neonothopanus nambi fungus belongs to the novel still undescribed protein family. The structure data for this protein is almost absent. A detailed study of the N. nambi luciferase properties is necessary for the improvement of analytical methods based on the fungal bioluminescent system. Here we present the positions of key amino acid residues and their effect on enzyme function described using bioinformatic and experimental approaches. These results are useful for further fungal luciferase structure determination.

Bioluminescence is a phenomenon of light emission resulting from oxidation of a substrate, luciferin, catalyzed by the enzyme luciferase. The fungus Neonothopanus nambi is the first eukaryotic organism with a fully deciphered bioluminescent system: the structure of luciferin was established, the luciferase gene was described, and intermediates and enzymes involved in the luciferin biosynthesis pathway were identified. One of the crucial reactions in this pathway is the formation of luciferin by hydroxylation of hispidin catalyzed by hispidin-3-hydroxylase (nnH3H). To fully understand the mechanism of action and substrate specificity of the enzyme, it is necessary to carry out structural studies of the molecule. To do that, it is necessary to develop a protocol for obtaining a highly purified and functionally active nnH3H in the appropriate quantities. We describe a robust approach to produce a soluble and enzymatically active nnH3H fused with SUMO and coexpressed with GroEL/ES chaperonin at low temperature in Escherichia coli. The yield of recombinant nnH3H achieved was 20 mg per 100 mL of bacterial culture. Additionally, we show for the first time that FAD is a cofactor of fungal hispidin-3-hydroxylase.

Morskie polihety Chaetopterus variopedatus (Renier) (semejstvo Chaetopteridae) — kosmopolity, predstavlyayushchie soboj vidovoj kompleks iz otdel'nyh populyacij-podvidov. Pri razdrazhenii chervi vypuskayut svetyashchiesya (460 nm) oblaka slizi, pri etom chasto yarko svetyatsya i ih parapodii. Na segodnyashnij den' po-prezhnemu ne yasno, kak imenno rabotaet biolyuminescentnaya sistema etih polihet. Ranee bylo vydvinuto predpolozhenie, chto lyuciferaza C. variopedatus mozhet byt' ispol'zovana dlya detekcii ferroptoza — nedavno otkrytogo puti programmiruemoj kletochnoj gibeli, vyzvannoj nakopleniem ionov dvuhvalentnogo zheleza. Cel'yu issledovaniya bylo vydelit' i oharakterizovat' lyuciferazy C. variopedatus, a takzhe sravnit' lyuciferazy C. variopedatus iz raznyh populyacij. Pri vydelenii otvetstvennogo za biolyuminescenciyu fermenta iz zamorozhennyh obrazcov brazil'skih C. variopedatus po usovershenstvovannoj metodike byli polucheny dve aktivnye lyuciferazy — L1 i L2. Predpolozhiv, chto odna iz ukazannyh lyuciferaz opredelyaet svechenie slizi, a drugaya — svechenie parapodij chervej, etu zhe metodiku primenili k razdel'nym obrazcam slizi i parapodij zhivyh dal'nevostochnyh C. variopedatus. Odnako ih sliz' okazalas' nesvetyashchejsya. Pokazano, chto funkciyu svecheniya parapodij polihet C. variopedatus obespechivaet lyuciferaza L2, tak kak ona obnaruzhena v obshchej biomasse brazil'skih polihet i v parapodiyah dal'nevostochnyh polihet. Svechenie slizi brazil'skih C. variopedatus obuslovleno funkcionirovaniem lyuciferazy L1, kotoraya otsutstvuet v slizi dal'nevostochnogo podvida. Nabor izoform lyuciferaz polihet C. variopedatus zavisit ot mesta ih obitaniya.

A key component of the recently described bioluminescent system of higher fungi is luciferase, a new class of proteins. The properties of fungal luciferase and their relationship with its structure are interesting both for improving autoluminescent systems already created on its basis and for creating new ones. Therefore, it is extremely important to understand the spatial structure of this protein. We have performed heterologous expression and purification of Neonothopanus nambi luciferase, obtained a protein suitable for subsequent crystallization, and also determined some biochemical properties of the recombinant luciferase.

This is the first study to obtain a high-purity luciferase from the fungus Neonothopanus nambi biomass that is suitable for subsequent sequencing.

The structure of fungal oxyluciferin was determined, the enzymatic bioluminescence reaction under substrate saturation conditions with discrete monitoring of formed products was conducted, and the structures of the end products of the reaction were established. On the basis of these studies, the scheme of oxyluciferin degradation to the end products was developed. The structure of fungal oxyluciferin was confirmed by counter synthesis.

Определили структуру оксилюциферина грибов, провели ферментативную реакцию биолюминесценции в условиях насыщения по субстрату с дискретным мониторингом образующихся продуктов и установили структуры конечных продуктов реакции. На основе этих исследований разработали схему деградации оксилюциферина до конечных продуктов. Структуру оксилюциферина грибов подтвердили встречным синтезом.

Multicolour labelling with fluorescent proteins is frequently used to differentially highlight specific structures in living systems. Labelling with fusion proteins is particularly demanding and is still problematic with the currently available palette of fluorescent proteins that emit in the red range due to unsuitable subcellular localization, protein-induced toxicity and low levels of labelling efficiency. Here we report a new monomeric red fluorescent protein, called FusionRed, which demonstrates both high efficiency in fusions and low toxicity in living cells and tissues.