Biology subjects

Trylska, J.

Publications and source records attributed to Trylska, J..

3 recordsLinked to original sources

bioRxiv2026

Crowder-specific modulation of hepatitis C virus NS3/4A protease activity and local structural dynamics

Macromolecular crowding modulates enzyme behavior in crowder- and protein-specific ways, yet its impact on viral proteases, which are often key therapeutic targets, remains unclear. Here, we investigated the hepatitis C virus NS3/4A protease under increasing concentrations of polyethylene glycols (PEGs), ficoll, dextran, and lysozyme using a fluorescence-based activity assay and intrinsic tryptophan fluorescence. PEGs reduced catalytic activity while leaving substrate binding largely unaffected or moderately enhanced. These effects were accompanied by a moderate tryptophan fluorescence spectral narrowing, consistent with reduced heterogeneity in local conformational environments. In contrast, ficoll enhanced catalytic efficiency despite stronger fluorescence quenching, indicating local structural changes that favored catalysis. Dextran and lysozyme inhibited protease activity through distinct kinetic patterns, likely reflecting differences in their size, shape, and chemical properties. Thermal analysis revealed crowder-specific local structural changes in NS3/4A without global unfolding up to 65{degrees}C, with differences in local stability and flexibility corroborating the observed kinetic effects. These findings demonstrate that macromolecular crowding modulates NS3/4A catalysis through crowder-specific effects on local structure.

biophysics↗

bioRxiv2025

Modulating backbone flexibility in hydroxamate siderophores for improved iron chelation and peptide nucleic acid (PNA) delivery into bacteria

Peptide nucleic acid (PNA) is a synthetic oligonucleotide analog with a peptide-based backbone that selectively binds with high affinity to natural nucleic acids. PNA is a valuable tool in antisense technology with potential antibacterial applications. However, PNA cannot penetrate bacterial cells alone. To address this, we explored iron chelators - siderophores - as PNA carriers. Bacteria acquire iron through siderophores, which are transported via specific TonB-dependent receptors in the bacterial envelope. Previously, we demonstrated that a synthetic hydroxamate-type siderophore (SL) exploited this transport system to deliver PNA into bacterial cells, achieving a gene-silencing effect. However, this transport was limited to an Escherichia coli mutant with continuous iron uptake, and was not observed in wild-type E. coli. In this study, we developed a new synthetic siderophore (SGLY) with glycine spacers between modified ornithine residues for enhanced flexibility and iron-binding. We also synthesized marine siderophore analogs (MGLY and MALA) inspired by natural moanachelins. Using circular dichroism spectroscopy, spectrophotometric assays, and molecular dynamics simulations, we confirmed iron binding. Growth recovery experiments showed SGLY recognition and internalization via the TonB-dependent transport system, likely using hydroxamate siderophore pathways. The MGLY and MALA siderophores showed lower growth promotion than SGLY, indicating less efficient internalization. Molecular docking revealed high affinity of SGLY for E. coli receptors involved in the uptake of hydroxamate siderophores. However, upon conjugation to PNA, all three siderophores effectively delivered PNA into E. coli cells. We confirmed PNA-mediated gene silencing using fluorescence measurements and confocal microscopy.

biochemistry↗

bioRxiv2022

Varying molecular interactions explain crowder-dependent enzyme function of a viral protease

Biochemical processes in cells, including enzyme-catalyzed reactions, occur in crowded conditions with various background macromolecules occupying up to 40% of cytoplasms volume. Viral enzymes in the host cell also encounter such crowded conditions as they often function at the endoplasmic reticulum membranes. We focus on an enzyme encoded by the hepatitis C virus, the NS3/4A protease, which is crucial for viral replication. We have previously found experimentally that synthetic crowders, polyethylene glycol (PEG) and branched polysucrose (Ficoll), differently affect the kinetic parameters of peptide hydrolysis catalyzed by NS3/4A. To gain understanding of the reasons for such behavior, we perform atomistic molecular dynamics simulations of NS3/4A in the presence of either PEG or Ficoll crowders and with and without the peptide substrates. We find that both crowder types make nanosecond long contacts with the protease and slow down its diffusion. However, they also affect the enzyme structural dynamics; crowders induce functionally relevant helical structures in the disordered parts of the protease cofactor, NS4A, with the PEG effect being more pronounced. Overall, PEG interactions with NS3/4A are slightly stronger but Ficoll forms more hydrogen bonds with NS3. The crowders also interact with substrates; we find that the substrate diffusion is reduced much more in the presence of PEG than Ficoll. However, contrary to NS3, the substrate interacts more strongly with Ficoll than with PEG crowders, with the substrate diffusion being similar to crowder diffusion. Importantly, crowders affect also the substrate-enzyme interactions. We observe that both PEG and Ficoll enhance the presence of substrates near the active site, especially near catalytic His57 but Ficoll crowders increase substrate binding more than PEG molecules. The presence of crowders also enhances the stability of Zn2+ ion coordination necessary for structural stability of NS3/4A enabling catalysis. AUTHOR SUMMARYEnzyme-catalyzed reactions in reality occur in the crowded environment of the cell. Therefore, viruses entering the host cells also encounter a crowded surrounding in which the viral enzymes are replicated. One such enzyme is the NS3/4A protease encoded by the hepatitis C virus. This enzyme is crucial for viral replication and is used as the therapeutic target for clinically approved drugs. To gain understanding of this enzyme function and explain our previous experiments on its in vitro activity, we performed atomistic molecular dynamics simulations in the presence of synthetic crowders (polyethylene glycol and polysucrose) mimicking the cellular crowd. Based on these simulations we describe in detail how and why these crowders affect the diffusion and structural dynamics of this enzyme and enzyme-substrate interactions. In fact, crowders enhance substrate binding, which may have vast consequences for its function in the host cell and drug-design.

bioinformatics↗