Biology subjects

Vacha, R.

Publications and source records attributed to Vacha, R..

16 recordsLinked to original sources

bioRxiv2026

Peptide Sequence Features that Reduce Membrane Pore Line Tension

Membrane pore stability is central to many processes involving membrane permeabilization, yet it remains unclear how the sequence of pore-localizing peptides can modify the energetics of the pore boundary. For large pores, the energetic cost associated with increasing pore size is described by the membrane line tension. Peptides capable of reducing line tension can therefore stabilize permeable membrane states, making their identification relevant for the design of membrane-active molecules. Here, we combine coarse-grained molecular dynamics simulations, free energy calculations, and evolutionary optimization algorithms to identify sequence features of -helical peptides that reduce membrane line tension. The best-performing peptides consistently showed an amphipathic organization with aromatic-rich termini, a hydrophobic/aromatic membrane-facing nonpolar face, and a negatively charged polar face. These sequence features promoted peptide localization at the pore rim close to the intact bilayer and orientation parallel to the membrane edge. This binding geometry reorganized lipids at the pore rim and efficiently reduced the exposure of their hydrophobic tails to water, thereby reducing the energetic cost of the pore boundary. The main sequence and mechanistic trends identified in coarse-grained simulations were reproduced in all-atom simulations. Together, these results link peptide sequence to pore-rim localization, lipid reorganization, and ultimately line-tension reduction, providing molecular design principles for $\alpha$-helical peptides that stabilize permeable membrane states.

biophysics↗

bioRxiv2026

Membrane Thickness Strain from Protein Inclusions: A Multiscale Simulation and X-Ray Scattering Study of Proteoliposomes

Integral membrane proteins remodel the surrounding lipid bilayer, but quantifying the resulting deformations and linking them to protein density in the membrane has remained challenging. Here, we introduce an integrative methodology that combines all-atom molecular dynamics (MD) simulations with multiscale small-angle X-ray scattering (SAXS) analysis to connect membrane strain to the protein/lipid ratio in proteoliposomes. Using outer membrane phospholipase A (OmpLA) reconstituted into lipid bilayers with both increased and decreased hydrophobic thickness, we systematically probe the effects of positive and negative hydrophobic mismatch. MD simulations demonstrate that OmpLA causes anisotropic, oscillatory thickness deformations extending up to eight times the radius of the first lipid shell surrounding the protein, yet the net change in average membrane thickness remains below 1%. Through our multiscale SAXS analysis, we quantitatively extract structural parameters, ranging from proteoliposome size to internal membrane architecture, using constrained Bayesian inference, with priors derived from MD findings. Specifically, we determine the protein/lipid molar ratio and average membrane strain, revealing excellent agreement between experiment and simulation. In thinner bilayers, substantial protein loss limits the analysis, highlighting the role of bilayer stability in sample preparation. Moreover, the predominance of OmpLA monomers in the thicker membranes is consistent with weak, membrane-mediated repulsive interactions between protein inclusions. Collectively, this integrative approach establishes a framework for quantifying protein-lipid interactions across molecular and mesoscale dimensions.

biophysics↗

bioRxiv2025

Quantifying (de)Mixing of Disordered Proteins in Molecular Dynamics Simulations

Biomolecular condensates control the spatial and temporal organization of cellular biochemistry. Their architectures often arise from complex, multicomponent mixtures governed by weak, multivalent interactions between intrinsically disordered regions (IDRs) of proteins. However, current approaches lack generalizable metrics to determine whether IDRs will mix or segregate within condensates. Here, we present a domain decomposition method (DDM) that accurately determines phase concentrations without identifying condensate interfaces. When tested against 399 IDR simulations, the DDM recovers dense- and dilute-phase concentrations within 1% agreement of established surface-based methods. By calculating spatial variances to map three-dimensional organization, this approach successfully distinguishes homogeneous mixing from surface enrichment, core--shell architectures, and complete segregation. Applying this continuous metric to 2,068 binary mixtures of 32 distinct IDRs, we demonstrate that physicochemical similarity strongly promotes mixing. Hydrophobic IDRs mixed promiscuously across all partner chemistries, whereas charged sequences mixed in a way highly sensitive to charge complementarity, forming the most stable condensates as net charge approached zero. Compared with experimental networks, our IDR-only simulations predicted the partitioning preferences for 10 of 13 proteins, with discrepancies accurately isolating cases where colocalization strictly requires RNA or folded domains. Quantifying mixing in this way provides a robust and reproducable method for predicting IDR interactomes and understanding the composition of complex cellular condensates.

biophysics↗

bioRxiv2025

gorder: Comprehensive tool for calculating lipid orderparameters from molecular simulations

Lipid order parameters are an important metric for quantifying the molecular structure of biological membranes. They can be derived from both molecular simulations and experimental measurements, enabling robust comparisons between the two. Although methods for calculating lipid order parameters from molecular dynamics simulations of membrane systems at various resolutions are well established, a comprehensive and user-friendly package for these calculations is lacking, which has even led some researchers to use tools that are known to perform the calculations incorrectly. To address this, we have developed gorder, an analysis tool capable of calculating lipid order parameters in atomistic, united-atom, and coarse-grained systems, compatible with any force field, and applicable to both planar and curved membrane geometries. gorder is designed to be fast and versatile, providing a unified solution for lipid order calculations. The tool is freely available from crates.io/crates/gorder and github.com/Ladme/gorder under the MIT License.

biophysics↗

bioRxiv2025

Mechanisms of transcription attenuation and condensation of RNA polymerase II by RECQ5

The elongation rates of RNA polymerase II (RNAPII) require precise control to prevent transcriptional stress, which can impede co-transcriptional pre-mRNA processing and contribute to many age- or disease-associated molecular changes (e.g., loss of proteostasis)1-5. Additionally mesoscale organization of transcription is thought to control the transcriptional rates6 and multiple factors have been reported to form biomolecular condensates and integrate RNAPII through the interaction with the C-terminal domain (CTD) of the largest subunit, RPB17-10. However, the structural organization of these condensates remains uncharacterized due to their small size and inherently dynamic nature. Here, we investigated the molecular mechanisms by which a general transcription factor - RECQ5 - associates with hyperphosphorylated RNAPII elongation complex (P-RNAPII EC) and controls translocation of RNAPII along genes. We combined biochemical reconstitution, electron cryomicroscopy, cryotomography, and coarse-grained simulations. We report two mechanisms by which RECQ5 modulates RNAPII transcription. At the atomic level, we demonstrate that RECQ5 uses the brake helix as a doorstop to control RNAPII translocation along DNA, attenuating transcription. At the mesoscale level, RECQ5 forms a condensate scaffold matrix, integrating P-RNAPII EC through a network of site-specific interactions, reinforcing the condensates structural integrity. Our integrative, multi-scale study provides insights into the structural basis of transcription attenuation and into the molecular architecture and biogenesis of a model RNAPII condensate.

biochemistry↗

bioRxiv2024

Development of a cell-permeable Biotin-HaloTag ligand to explore functional differences between protein variants across cellular generations

HaloTag technology represents a versatile tool for studying proteins. Fluorescent HaloTag ligands employed in sequential labeling led to the discovery of distinct protein variants for histones, cohesins, and MCM complexes. Nonetheless, an efficient biochemical approach to separate the distinct protein variants to study their biological functions is missing. Principally being a gap in technology, the HaloTag toolbox lacks affinity ligands displaying good cell permeability and efficient affinity capture. Here, we describe the design, synthesis, and validation of a new cell-permeable Biotin-HaloTag ligand, which allows rapid labeling of Halo-tagged proteins in live cells and their efficient separation using streptavidin pull-down. Our work outlines how to use the herein-developed affinity ligand in sequential labeling to biochemically separate distinct protein variants and study their biological properties. The approach holds immense potential for addressing fundamental questions concerning essential cellular processes, including genome duplication and chromatin maintenance.

cell biology↗

bioRxiv2024

Role of Divalent Ions in Membrane Models of Polymyxin-Sensitive and Resistant Gram-Negative Bacteria

Polymyxins, critical last-resort antibiotics, impact the distribution of membrane-bound divalent cations in the outer membrane of Gram-negative bacteria. We employed atomistic molecular dynamics simulations to model the effect of displacing these ions. Two polymyxin-sensitive and two polymyxin-resistant models of the outer membrane of Salmonella enterica were investigated. First, we found that the removal of all calcium ions induces a global stress on the model membranes, leading to substantial membrane restructuring. Next, we used enhanced sampling methods to explore the effects of localized stress by displacing membrane-bound ions. Our findings indicate that creating defects in the membrane-bound ion network facilitates polymyxin permeation. Additionally, our study of polymyxin-resistant mutations revealed that divalent ions in resistant model membranes are less likely to be displaced, potentially contributing to the increased resistance associated with these mutations. Lastly, we compared results from all-atom molecular dynamics simulations with coarse-grained simulations, demonstrating that the choice of force field significantly influences the behavior of membrane-bound ions under stress.

biophysics↗

bioRxiv2023

Enhanced Diffusion through Multivalency

In multivalent systems, multiple ligands from one entity simultaneously bind to multiple receptors on another entity. These interactions are of crucial significance in a wide range of biological and technological mechanisms, encompassing selectivity, host recognition, viral penetration, therapeutic delivery, as well as the adhesion phenomena found in cells, polymers, and nanoparticles. In this study, we used computer simulations to investigate 1D and 2D diffusion of adsorbed particles with varying valency but with the same overall affinity to the host. We demonstrate a remarkable diffusion acceleration for particles with increasing valency. Non-diffusing monovalent particle can attain almost unrestricted diffusion when becoming multivalent while retaining its affinity for the host tether or surface. Moreover, diffusion of multivalent particles with rigid ligand distribution can be controlled by patterned host receptors. Our results have practical implications for the design of fast-diffusing particles that maintain a strong affinity for target surfaces or molecules. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/558647v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@ef72d1org.highwire.dtl.DTLVardef@5b22e7org.highwire.dtl.DTLVardef@150868borg.highwire.dtl.DTLVardef@1b1364e_HPS_FORMAT_FIGEXP M_FIG C_FIG SignificanceWe investigated how the number of binding sites (referred to as valency) on particles or entities impacts their movement when attached to surfaces or filaments. Valency can be understood as how many "hands" a particle has to grip the surface. Surprisingly, particles with more "hands" move faster if they hold onto the surface with the same strength. Furthermore, the motion of these particles can be controlled by designing surfaces with specific patterns that the "hands" can grasp. This means that we can design particles that move rapidly while remaining attached to the desired locations. These findings hold promise for applications like drug delivery and materials technology, and for understanding biological processes.

biophysics↗

bioRxiv2023

Valency of Ligand-Receptor Binding Ignored by Pair Potentials

Molecular dynamics simulations have been crucial for investigating the dynamics of nanoparticle uptake by cell membranes via ligand-receptor interactions. Use of coarsegrained models has enabled evaluation of the effects of nanoparticle size, shape, ligand distribution on nanoparticles surface, or used thoroughly in the past decade, where a percentage of lipid heads, receptors, are attracted to sites on the nanoparticle surface, ligands. However, when pair-potentials are used to represent ligand-receptor interactions, the number of receptors interacting with one ligand, valency, may vary. We demonstrate that the curvature of a nanoparticle, strength of ligand-receptor interactions, and ligand or receptor concentration change the valency - ranging from 3.4 to 5.1 in this study. Such change in valency can create inaccurate comparisons between nanoparticles, or even result in the uptake of smaller nanoparticles than would be expected. To rectify this inconsistency we propose the adoption of a model based on bond-formation and use it to determine the extent to which previous studies may have been effected. This work recommends avoiding pair-potentials for modeling ligandreceptor interactions to ensure methodological consistency in nanoparticle studies. TOC GraphicA rendering of a ligand coated nanoparticle coming into contact with a lipid bilayer membrane. The receptor in the membrane is highlighted for clarity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/557452v3_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@10730a2org.highwire.dtl.DTLVardef@109f0b9org.highwire.dtl.DTLVardef@19b0d80org.highwire.dtl.DTLVardef@93557a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Insertases Scramble Lipids: Molecular Simulations of MTCH2

Scramblases play a pivotal role in facilitating bidirectional lipid transport across cell membranes, thereby influencing lipid metabolism, membrane homeostasis, and cellular signaling. MTCH2, a mitochondrial outer membrane protein insertase, has a membrane-spanning hydrophilic groove resembling those that form the lipid transit pathway in known scramblases. Employing both coarse-grained and atomistic molecular dynamics simulations, we show that MTCH2 significantly reduces the free energy barrier for lipid movement along the groove and therefore can indeed function as a scramblase. Notably, the scrambling rate of MTCH2 in silico is similar to that of VDAC, a recently discovered scramblase of the outer mitochondrial membrane, suggesting a potential complementary physiological role for these mitochondrial proteins. Finally, our findings suggest that other insertases which possess a hydrophilic path across the membrane like MTCH2, can also function as scramblases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/553169v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@e78668org.highwire.dtl.DTLVardef@292ce0org.highwire.dtl.DTLVardef@1413077org.highwire.dtl.DTLVardef@d4e29b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIScrambling activity of MTCH2 identified using computer simulations C_LIO_LIMTCH2 may act redundantly with VDAC as outer mitochondrial membrane scram-blase C_LIO_LIInsertases and scramblases may share a common functional mechanism C_LI

biophysics↗

bioRxiv2023

Counterintuitive Binding of Phosphorylated DEP Domain from Dishevelled Protein to Negatively Charged Membranes

To accomplish its role of signaling hub in all Wnt signaling pathways, Dishevelled (DVL) protein needs to dynamically relocalize to the inner leaflet of the cellular plasma membrane (PM). Combined experimental and computational evidence showed that the binding of DVL to the PM is mainly driven by the electrostatic attraction between a stretch of positively charged amino acids located on the C-terminal DEP domain of DVL and anionic phospholipid species, with a striking preference for phosphatidic acid (PA). Here, by means of computational simulations and QCM-D experiments, we demonstrate that four recently identified phosphorylation sites on DEP domain, alter the electrostatic potential of the membrane binding interface, but do not prevent the recruitment to anionic membranes. On the contrary, the phosphorylated residues are involved in hydrogen bond and ion-mediated interactions with the lipid headgroup of PA. Our results suggest that the effect of phosphorylation on protein-membrane association could be counterintuitive and sensitive to changes in the local environment including specific lipids, salts, and pH. SIGNIFICANCEPhosphorylation regulates the cellular activity and localization of many peripheral proteins by, among others, decreasing the affinity for negatively charged membranes. Here, we report how phosphorylation affects the membrane interaction of DEP domain from Dishevelled protein, the intracellular signaling hub in Wnt pathways. We found that despite the negative charge induced by phosphorylation, DEP domain was steadily adsorbed to the surface of negatively charged PA-rich membranes, due to a dense network of cation-mediated interactions and hydrogen bonds.

biophysics↗

bioRxiv2022

Mitochondrial phospholipid import mediated by VDAC, a dimeric beta barrel scramblase

Mitochondria are double-membrane-bounded organelles that depend critically on phospholipids supplied by the endoplasmic reticulum. These lipids must cross the outer membrane to support mitochondrial function, but how they do this is unclear. We identified the voltage-dependent ion channel (VDAC), an abundant outer membrane protein, as a scramblase-type lipid transporter that catalyzes lipid entry. On reconstitution into membrane vesicles, dimers of human VDAC1 and VDAC2 catalyze rapid transbilayer translocation of phospholipids by a mechanism that is unrelated to their channel activity. Coarse-grained molecular dynamics simulations of VDAC1 reveal that lipid scrambling occurs at a specific dimer interface where polar residues induce large water defects and bilayer thinning. The rate of phospholipid import into yeast mitochondria is an order of magnitude lower in the absence of VDAC homologs, indicating that VDACs provide the main pathway for lipid entry. Thus, VDAC isoforms, members of a superfamily of beta barrel proteins, moonlight as a new class of phospholipid scramblases - distinct from alpha-helical scramblase proteins - that act by an unprecedented mechanism to import lipids into mitochondria.

biochemistry↗

bioRxiv2022

De novo design of peptides that form transmembrane barrel pores killing antibiotic resistant bacteria

De novo design of peptides that self-assemble into transmembrane barrel-like nanopore structures is challenging due to the complexity of several competing interactions involving peptides, lipids, water, and ions. Here, we develop a computational approach for the de novo design of -helical peptides that self-assemble into stable and large transmembrane barrel pores with a central nano-sized functional channel. We address the lack of existing design guidelines for the de novo pore-forming peptides and propose 52 sequence patterns, each of which can be tailored for different applications using the identified role of its residues. Atomic force microscopy, channel electrical recording, leakage of small fluorescent molecule and transport of macromolecule experiments confirm that the designed peptides form stable, large, and functional barrel-shaped nanopores in model membranes. The custom-designed peptides act as potent antimicrobial agents able to kill even antibiotic-resistant ESKAPE bacteria at micromolar concentrations, while exhibiting low toxicity to human cells. Peptides and their assembled nanopore structures can be similarly fine-tuned for other medical and biotechnological applications.

biophysics↗

bioRxiv2019

Magainin 2 and PGLa in Bacterial Membrane Mimics II: Membrane Fusion and Sponge Phase Formation

We studied the synergistic mechanism of equimolar mixtures of magainin 2 (MG2a) and PGLa in phosphatidylethanolamine/phosphatidylglycerol mimics of Gram-negative cytoplasmic membranes. In a preceding paper [Pachler et al., Biophys. J. 2019 xxx], we reported on the early onset of parallel heterodimer formation of the two antimicrobial peptides already at low concentrations and the resulting defect formation in membranes. Here, we focus on the structures of the peptide/lipid aggregates occurring in the synergistic regime at elevated peptide concentrations. Using a combination of calorimetric, scattering, electron microscopic and in silico techniques, we demonstrate that the two peptides, even if applied individually, transform originally large unilamellar vesicles into multilamellar vesicles, with a collapsed interbilayer spacing resulting from peptide induced adhesion. Interestingly, the adhesion does not lead to a peptide induced lipid separation of charged and charge neutral species. In addition to this behavior, equimolar mixtures of MG2a and PGLa formed surface-aligned fibril-like structures, which induced adhesion zones between the membranes and the formation of transient fusion stalks in molecular dynamics simulations and a coexisting sponge phase observed by small-angle X-ray scattering. The previously reported increased leakage of lipid vesicles of identical composition in the presence of MG2a/PGLa mixtures is therefore related to a peptide-induced cross-linking of bilayers.\n\nSTATEMENT OF SIGNIFICANCEWe demonstrate that the synergistic activity of the antimicrobial peptides MG2a and PGLa correlates to the formation of surface-aligned fibril-like peptide aggregates, which cause membrane adhesion, fusion and finally the formation of a sponge phase.

biophysics↗

bioRxiv2019

Magainin 2 and PGLa in Bacterial Membrane Mimics I: Peptide-Peptide and Lipid-Peptide Interactions

We addressed the onset of synergistic activity of the two well-studied antimicrobial peptides magainin 2 (MG2a) and PGLa using lipid-only mimics of Gram-negative cytoplasmic membranes. Specifically, we coupled a joint analysis of small-angle X-ray and neutron scattering experiments on fully hydrated lipid vesicles in the presence of MG2a and L18W-PGLa to all-atom and coarse-grained molecular dynamics simulations. In agreement with previous studies both peptides, as well as their equimolar mixture, were found to remain in a surface-aligned topology upon membrane insertion and to induce significant membrane perturbation as evidenced by membrane thinning and hydrocarbon order parameter changes in the vicinity of the inserted peptide. These effects were particularly pronounced for the so called synergistic mixture of 1:1 (mol/mol) L18W-PGLa/MG2a and cannot be accounted for by a linear combination of the membrane perturbations of two peptides individually. Our data are consistent with parallel heterodimers forming at much lower concentrations than previously considered, but which do not induce a synergistic leakage of dyes. Our simulations further show that the heterodimers interact via salt bridges and hydrophobic forces, which apparently makes them more stable than putatively formed antiparallel L18W-PGLa and MG2a homodimers. Moreover, dimerization of L18W-PGLa and MG2a leads to a relocation of the peptides within the lipid headgroup regime as compared to the individual peptides. The early onset of dimerization of L18W-PGLa and MG2a at low peptide concentrations consequently appears to be key to their synergistic dye-releasing activity from lipid vesicles at high concentrations.\n\nSTATEMENT OF SIGNIFICANCEWe demonstrate that specific interactions of the antimicrobial peptides MG2a and PGLa with each other in POPE/POPG bilayers lead to the formation of surface-aligned parallel dimers, which provide already at low peptide concentrations the nucleus for the peptides well-known synergistic activity.

biophysics↗

bioRxiv2019

Effect of helical kink in antimicrobial peptides on membrane pore formation

Every cell is protected by a semipermeable membrane. Peptides with the right properties, e.g. Antimicrobial peptides (AMPs), can disrupt this protective barrier by formation of leaky pores. Unfortunately, matching peptide properties with their ability to selectively form pores in bacterial membranes remains elusive. In particular, the proline/glycine kink in helical peptides was reported to both increase and decrease antimicrobial activity. We used computer simulations and fluorescence experiments to show that a kink in helices affects the formation of membrane pores by stabilizing toroidal pores but disrupting barrel-stave pores. The position of the proline/glycine kink in the sequence further controls the specific structure of toroidal pore. Moreover, we demonstrate that two helical peptides can form a kink-like connection with similar behavior as one long helical peptide with a kink. The provided molecular-level insight can be utilized for design and modification of pore forming antibacterial peptides or toxins.

biophysics↗