Microplastics introduced into freshwater environments create novel surfaces that select for specific microbial colonizers and exclude others. In urban rivers, these biofilms can act as reservoirs of antimicrobial resistance and contain potential enzymes for polymer degradation. We studied microbial communities associated with microplastics in the Setun River and examined how their composition changes during laboratory enrichment on plastic substrates. Native river specimen and cultures enriched on low-density polyethylene (LDPE) and polycaprolactone (PCL) were analyzed using mWGS and full-length 16S rRNA nanopore sequencing. Enrichment led to a pronounced shift toward nearly monoculture of Bacillota, more specifically Bacillus cereus, while native plastisphere communities were dominated by Pseudomonadota. Microscopy revealed clear degradation of PCL but not LDPE, and functional screening of native metagenomes uncovered a diverse resistome, including oqxAB efflux operons, mcr-3-like phosphoethanolamine transferases, various beta-lactamases, and class 1 integron genes, demonstrating that the Setun River plastisphere already contained clinically relevant AMR determinants. These findings suggest that certain bacteria such as Bacillus cereus can thrive and dominate on plastic surfaces in urban rivers, while many other taxa cannot persist there, highlighting that microplastics strongly reshape plastisphere communities and emphasize the role of river-borne microplastics as potential vectors of antibiotic resistance.

QM/MM calculations were performed to determine the energy differences between the green and red states of AzamiGreen fluorescent protein variants. The contributions of the resonance P- and I-forms of the chromophore were analyzed for a series of five proteins. A relationship was demonstrated between the contribution of the phenolate anionic form and the stabilization of the red state of the chromophore.

It was shown previously that mycobacteria (Mycobacterium smegmatis and Mycobacterium tuberculosis) accumulate tetramethyl ether of coproporphyrin III (TMC) during transition to dormant state. However, the enzymes involved in TMC synthesis remained unknown. Here we have identified a new M. smegmatis methyltransferase MSMEG_0614 (CPmtA) that catalyzes the S-adenosyl-L-methionine-dependent conversion of coproporphyrin (CP) into TMC through sequential methylation of its carboxyl groups. Overexpression of MSMEG_0614 in both M. smegmatis and M. tuberculosis leads to increase in intracellular TMC levels and reduction of bacterial respiratory activity, linking this enzymatic pathway to dormant state transition in mycobacteria. Structural-dynamic modeling reveals a unique “carousel” mechanism in which the orientation of CP alternates within the enzyme's active site to methylate its carboxyl groups sequentially. Mutagenesis based on computational predictions validates the catalytic mechanism. In addition, simulations demonstrate that TMC, unlike CP, integrates into a model phospholipid membrane, altering its properties, which possibly are important for transition to dormancy.

Nucleophilic substitution at phosphorus centers is among the most widely spread biochemical reactions of natural and artificial organophosphates. These reactions can occur via dissociative and associative mechanisms, depending on a particular enzyme and substrate. The most straightforward yet expensive way to determine the mechanism is to calculate the energy profile for the entire reaction. In this study, we analyzed a set of 15 enzymes that cleave P-O bonds in different organophosphates using QM/MM methods on potential energy surfaces and in MD simulations, combined with subsequent electron density analysis. We demonstrate that the structure and electron density features of the reaction region in the enzyme-substrate complex determine the mechanism of the subsequent reaction. The cleaving P-OLG bond is elongated in systems that undergo chemical reactions via a dissociative mechanism compared with the associative one. Importantly, the bond length distribution remains the same upon changing the DFT functional in simulations. Likely, the P-OLG bond length is mostly determined by the protein environment rather than the nature of the nucleophile: a virtual change of the neutral water molecule to the hydroxide anion does not shift the distance distribution. The cleaving P-OLG bond order, calculated from electron density, is also larger for systems operating via an associative pathway. Both distances and bond orders change continuously, and it is not evident what the border value that discriminates different mechanisms is. Therefore, we propose a binary classifier that relies on the Laplacian of the electron density in the cleaving P-OLG bond region. Electron density concentration is observed only for systems that perform reactions via an associative pathway. This criterion was tested on three adenylate kinases. Despite their structural differences, we obtained the same dissociative reaction mechanism type, which further proves the reliability of the suggested criterion.

Layered hybrid halide compounds offer promising functional properties, particularly tunable band gaps, conductivity, light harvesting thus making them prospective for applications in photovoltaics and optoelectronics. This study exemplifies an approach of predicting band gaps using machine learning models enhanced by invariant topological representations of these materials using the atom-specific persistent homology method in order to facilitate the discovery and design of new hybrid halide materials with tailored electronic properties.

We present the results of experimental and theoretical studies of excited state dynamics of flavin adenine dinucleotide (FAD) in water-methanol and water-ethanol mixtures as a function of alcohol concentration. The experimental studies have been carried out by recording time-resolved polarized fluorescence in FAD after excitation with short laser pulses using the time-correlated single photon counting method. The results obtained have shown that in aqueous solution fluorescence decay in FAD could be presented as a sum of four exponents with decay times of 20 ps, 210 ps, 2.70 ns, and 3.85 ns. Addition of methanol, or ethanol only insignificantly affected the decay time values, however caused dramatic changes in the contributions of the exponents to the fluorescence decay signal. Molecular dynamics (MD) simulations and QM/MM calculations in water-methanol and water-ethanol mixtures have been carried out and revealed the existence of three distinct conformation groups of FAD: Stack I, Stack III, and Open, which differ by mutual positions of the adenine and isoalloxazine rings and interaction between them. A model has been developed for elucidation of the excited state dynamics in FAD and of the nature of the heterogeneity of the recorded fluorescence decay times. The model classifies several relaxation channels in FAD after excitation by short laser pulses and suggests that the sub-nanosecond decay times of 20 ps and 210 ps both reflect fast fluorescence quenching due to electron transfer reactions in the vicinity of a conical intersection in the Stack III and Stack I conformations of FAD. The Stack I conformation is mostly stabilized by intramolecular forces due to pi-stacking interactions between the adenine and isoalloxazine rings and internal hydrogen bonds, while the Stack III conformation is stabilized to a large extent by hydrogen bonds with external water molecules. It was also suggested that the nanosecond decay times of 2.70 and 3.85 ns were governed mostly by relatively weak non-radiative decay channels from the bottom of the lowest excited electronic state either via direct relaxation to the ground state, or via tunneling to a redox-pair excited state through potential barrier. The decay time of 2.70 ns was shown to refer mainly to folded conformations and the decay time of 3.85 ns to open conformations.

Fluorescent proteins find application as biocompatible, genetically encoded labels for visualization of living organisms tissues. Green fluorescent proteins (GFPs) are the most diverse, but proteins with red fluorescence have advantages, such as lower phototoxicity and better penetration into biological tissues. A promising approach is to obtain red fluorescent proteins (RFPs) from GFPs by introducing mutations that stabilize the oxidized chromophore state with an extended conjugated π-system. However, to date this remains a non-trivial task and experimental developments are carried out mainly by random mutagenesis. Development of descriptors obtained in molecular modeling can rationalize this field. Herein, we rely on experimental data on the AzamiGreen fluorescent protein and its variants that are oxidized to the red form. We perform classical molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) simulations to determine structural and dynamic features that govern oxidation. We demonstrate that the red state is predominantly stabilized by interactions of polar lysine residues with chromophore oxygen atoms. Dynamic network analysis demonstrates that in red fluorescent proteins the chromophore motions are correlated with the movement of surrounding protein side chains to a higher extent than in green variants. The presence of different resonance forms of the chromophore determines the fluorescence band maximum value: a decrease in the phenolate form population leads to the red shift.

Antibodies against low-molecular-weight compounds exhibit cross-reactivities (CRs) with their structural analogs, varying by orders of magnitude for different substances. This variability limits the informativeness of antibody applications as analytical reagents and for other aims when samples contain several members of the same family, their derivatives, or partial degradation products. Therefore, there is a demand to find some criteria for understanding the relationships between the structural characteristics of antigens of a given chemical class and their immunochemical activity. This study presents an experimental and theoretical investigation of the properties of a monoclonal antibody (MAb) against the S-stereoisomer of gatifloxacin, a member of the widely used (fluoro)quinolone (FQ) family of antibiotics, characterized by high structural diversity. The aim was to determine FQs that form complexes with MAb and suggest a methodology to predict their CRs in silico. For this, the interaction of MAb with 26 FQs was studied using the enzyme-linked immunosorbent assay and presented as CR values to the target antigen. The most pronounced CRs were observed for lomefloxacin, sarafloxacin, and ciprofloxacin. Molecular dynamics (MD) simulations were performed to identify differences in analyte interactions at the MAb antigen-binding site, which determines binding affinity. It has been shown that molecular docking fails to discriminate cross-reactive from non-cross-reactive compounds because FQs have similar cores. Therefore, advanced analysis of MD trajectories was carried out. It allowed for clarification of the dynamic features of analyte-antibody interactions responsible for binding. It was shown by the dynamical network analysis that the sum of betweenness centrality between a node corresponding to the quinolone ring and nodes representing MAb amino acids is higher for cross-reactive haptens. The found regularities can be transferred to other analyte-antibody systems as a binary classifier that discriminates cross-reactive and non-cross-reactive compounds.

In nanopore sequencing, especially in SELEX-based aptamer discovery, the correct ligation of artificial sequences (primers, adapters, barcodes) is crucial for library quality. Errors at this stage can lead to misidentification of sequences and loss of valuable information. Existing quality control tools lack focused capabilities to assess the positioning and prevalence of these artificial sequences. NanoporeInspect is a web-based tool designed to fill this gap by providing targeted insights into ligation efficacy and systematic biases within sequencing data. NanoporeInspect operates as a user-friendly, web-based platform that leverages a modern software stack with Flask, Celery and Redis to handle scalable and asynchronous task processing, and Plotly to deliver interactive visualizations. Evaluation of NanoporeInspect on various nanopore datasets demonstrated its effectiveness in discerning differences in ligation quality. Libraries with inefficient ligation showed irregular adapter and barcode distributions, indicating preparation issues, while high-quality libraries displayed uniform patterns, reflecting effective ligation.

Nitrocefin hydrolysis by metallo-beta-lactamase is an important model chemical reaction mimicking cephalosporin antibiotic inactivation. Due to the specific chromogenic properties of the nitrocefin, transient kinetic data for this reaction is available. Despite its importance in the understanding of the reaction mechanism, these data can be utilised to verify benchmark calculations. This reaction is complicated from the computational viewpoint as the active site carries two double charged cations and therefore is highly polarised; nucleophilic attack and formation of the electrophilic site should be properly described. We calculate Gibbs-free energy profiles of three chemical steps comprising the entire reaction at the QM(DFT)/MM molecular dynamics level. We compare results obtained with three hybrid functionals differing in the contribution of the exact Hartree-Fock exchange, B3LYP-D3, PBE0-D3 and BHHLYP-D3. Among them, only calculations performed at the QM(PBE0-D3)/MM level were able to properly describe the intermediate accumulation and the limiting step. Laplacian of electron density maps clarify the influence of the computational protocol on the electrophilic site formation and covalent bond polarisation.

An interplay between the structural and physicochemical properties of the monoheme cytochromes of type c has been extensively studied. However new proteins belonging to this diverse family continue to reveal some novel and unique features. Here, we present the 1.15 Å structure of the low-potential cytochrome c546/556 from the bacterium Thioalkalivibrio paradoxus ARh1, which exhibits the prominent splitting of the Q bands in UV-visible spectra even at room temperature. The data obtained suggest that two conformations of the propionate 7 of the heme are responsible for the splitting of the Q bands. We propose that the degree of the splitting of the Q bands is correlated with the conformational lability of the heme propionates.

The Long Interspersed Element-1 (L1) retrotransposon is an ancient genetic parasite that comprises a significant part of the human genome. ORF2p is a multifunctional enzyme with endonuclease (EN) and reverse transcriptase (RT) activities that mediate target-primed reverse transcription of RNA into DNA. Structural studies of LINE-1 ORF2p consistently show a single Mg2+ cation in the reverse transcriptase active site, conflicting with the common DNA polymerase mechanism which involves two divalent cations. We explored a reaction pathway of the DNA elongation based on the recent high-resolution ternary complex structure of the ORF2p. The combined quantum and molecular mechanics approach at the QM (PBE0-D3/6-31G**)/MM (CHARMM) level is employed for biased umbrella sampling molecular dynamics simulations followed by umbrella integration utilized to obtain the free energy profile. The nucleotidyl transfer reaction proceeds in a single step with a free energy barrier of 15.1 ± 0.8 kcal/mol, and 7.8 ± 1.2 kcal/mol product stabilization relative to reagents. Concerted nucleophilic attack by DNA O3′ and proton transfer to Asp703 occur without a second catalytic metal ion. Estimated rate constant ∼60 s−1 aligns with RT kinetics, while analysis of the Laplacian of the electron density along the cleaving P-O bond identifies a dissociative mechanism.

Recent developments in computer technologies, software and methods have made molecular modeling a powerful tool in experimental studies of biomolecular systems, and in their rational modification [...]

Oxydifficidin is a natural polyketide antibiotic that has long been recognized as a ribosome-targeting agent that inhibits protein synthesis. In this paper, we describe Bacillus velezensis strain EV17 and compare its complete genome sequence with that of the previously characterized B. velezensis strain K-3618 and the difficidin biosynthetic gene cluster (BGC) combined with mass spectrometry to elucidate the production of oxydifficidin by these strains. Toeprinting and small fluorescent peptide assays showed that isolated oxydifficidin induces a generalized inhibition of translation at every step of elongation in protein biosynthesis. In previous studies, it has been demonstrated that oxydifficidin targets bL12 protein. Although spontaneous mutations conferring resistance to oxydifficidin in ribosomal protein bL12 located relatively close to the thiostrepton binding site on uL11, our data show that oxydifficidin binding does not interfere with thiostrepton, thereby refining previous findings about its putative ribosomal target. We are the first to show that this compound does not affect eukaryotic translation and has two orders of magnitude lower effect on eukaryotic cells compared to bacteria. These facts are important to further investigate its potential as a bioprotectant against phytopathogens or even as a therapeutic agent.

The CopC proteins are periplasmic copper binding proteins involved in bacterial copper homeostasis. One of the supposed functions of СopСs is to deliver copper ions for incorporation into the active sites of copper-containing oxidoreductases. In the sulfur oxidizing bacterium Thiolkalivibrio paradoxus, the genes encoding the CopCD proteins are located near the gene encoding the enzyme thiocyanate dehydrogenase (TcDH) containing three copper ions in the active site. The biochemical characterization demonstrated that CopC from Tv. paradoxus (tpCopC) has a high affinity for copper ions in both oxidation states (log KD = -16.3 ± 0.6 for Cu(II) and -11.1 ± 0.2 for Cu(I)). The protein Cu(II)-tpCopC forms a transient complex with TcDH, in which a copper ion could be transferred from tpCopC to the active site of TcDH. In the absence of a reducing agent, the transfer of 0.5 ± 0.2 copper ions is observed; under reducing conditions, the transfer of 2.4 ± 0.1 copper ions takes place followed by the activation of TcDH. Thus, CopC can act as a metallochaperone, providing the incorporation of copper ions into the active site of TcDH. The mechanism is proposed for the copper ion transfer from Cu(II)-tpCopC to TcDH through the intermediate reduction to form Cu(I)-tpCopC.

Fluorogen-activating proteins are powerful molecular tools for microscopy, including functional imaging. These proteins serve as an alternative to GFP-like proteins, as they do not require oxygen for chromophore maturation. However, the restricted selectivity of proteins to chromophores, combined with the limited number of spectral channels of conventional fluorescent microscopes, hinders the development of multicolor synthetic dyes. Additionally, the poor cell and tissue permeability of synthetic chromophores further limits their utility. In this work, we address these challenges by combining time-resolved methods with the rational design of the UnaG protein, which utilizes bilirubin as a natural chromophore. To turn UnaG into a palette of probes for fluorescence lifetime imaging microscopy (FLIM), we solved two practical problems: first, we determined the limits of bilirubin lifetime variations in response to changes in the protein structure and, second, we determined what minimal structural changes can be reliably distinguished by lifetime analysis in cellula. Combining classical point mutagenesis and the translational introduction of noncanonical amino acids, we generated UnaG with fluorescence lifetimes ranging from hundreds of picoseconds to nanoseconds. We explored the potential for further modification of the UnaG protein matrix to optimize spectral and temporal characteristics of bilirubin fluorescence and its quantitative detection through time-resolved approaches.

The active sites of enzymes are able to activate substrates and perform chemical reactions that cannot occur in solutions. We focus on the hydrolysis reactions catalyzed by enzymes and initiated by the nucleophilic attack of the substrate’s carbonyl carbon atom. From an electronic structure standpoint, substrate activation can be characterized in terms of the Laplacian of the electron density. This is a simple and easily visible imaging technique that allows one to “visualize” the electrophilic site on the carbonyl carbon atom, which occurs only in the activated species. The efficiency of substrate activation by the enzymes can be quantified from the ratio of reactive and nonreactive states derived from the molecular dynamics trajectories executed with quantum mechanics/molecular mechanics potentials. We propose a neural network that assigns the species to reactive and nonreactive ones using the Laplacian of electron density maps. The neural network is trained on the cysteine protease enzyme-substrate complexes, and successfully validated on the zinc-containing hydrolase, thus showing a wide range of applications using the proposed approach.

Nanopore sequencing is a promising technology for reading long fragments of DNA and RNA. This paper reviews modern algorithms and software tools used for the analysis of nanopore sequencing data in genomics, metagenomics, and epigenomics. The article describes the algorithms behind the most widely used software tools, highlighting their strengths and limitations. The presented review is useful for researchers working with nanopore sequencing data, as well as for developers of new methods for processing bioinformatics data.

ORF2p (open reading frame 2 protein) is a multifunctional multidomain enzyme that demonstrates both reverse transcriptase and endonuclease activities and is associated with the pathophysiology of cancer. The 3D structure of the entire seven-domain ORF2p complex was revealed with the recent achievements in structural studies. The different arrangements of the CTD (carboxy-terminal domain) and tower domains were identified as the "closed-ring" and "open-ring" conformations, which differed by the hairpin position of the tower domain, but the structural diversity of these complexes has the potential to be more extensive. To study this, we performed sub-microsecond all-atom molecular dynamics simulations of the entire ORF2p complex with different starting configurations. The obtained molecular dynamic trajectories frames were assigned to several clusters following the dimension reduction to three principal components of the 1275 distances feature matrix. Five and six clusters were obtained for the "open" and "closed" ring models, respectively. While the fingers-palm-thumb core retains its rigid configuration during the MD (molecular dynamics) simulations, all other domains display the complicated dynamic behavior not observed in the experimental structures. The EN (endonuclease) and CTD domains display significant translations and rotations while their internal structures stay rigid. The CTD domain can either form strong contacts with the tower or be far apart from it for both formal "open" and "closed" ring states because the tower hairpin position is not the only determining factor of the protein complex configuration. While only the "thumb up" conformation is observed in all the trajectories, the active site can be obstructed by the movement of the CTD domain. Thus, molecular modeling and machine learning techniques provide valuable insights into the dynamical behavior of the ORF2p complex, which is hard to uncover with experimental methods, given the complexity and size of the object.

Recent achievements in molecular modeling of reaction mechanisms of the enzymatic ATP conversion to ADP or cAMP are discussed. Both of these reactions are initiated by the nucleophilic attack of an oxygen atom, but the P–O bridging bond cleavage occurs via different mechanisms, dissociative and associative. These mechanisms differ in the order of formation and cleavage of P–O bonds. For ATP ases, the dissociative mechanism is assumed, whereas ATP conversion to the cAMP occurs via associative mechanism. We suggest a novel approach based on the molecular dynamics simulations with combined quantum mechanics/molecular mechanics potentials of the enzyme–substrate complexes that can discriminate dissociative and associative reaction pathways by analysis of length distributions of the cleaving and forming P–O bonds.