Biology subjects

Schäfer, L. V.

Publications and source records attributed to Schäfer, L. V..

15 recordsLinked to original sources

bioRxiv2026

Light Martini water accelerates sampling in coarse-grained molecular dynamics simulations

Molecular dynamics (MD) simulations of slow biomolecular processes, such as exploration of the conformational ensembles of intrinsically disordered proteins (IDPs), are computationally demanding. Although coarse-grained (CG) models can substantially speed up the simulations compared to all-atom MD, the sampling challenge can still be significant for large systems and long time scales. Here, we present light Martini water, a low-viscosity water model that accelerates sampling in MD simulations with the Martini CG force field. We systematically reduced the mass of the Martini water beads and verified stable, accurate integration of the equations of motion with 20 fs time steps, as typically used in Martini simulations. Light Martini water has a reduced mass of 20 amu (compared to 72 amu in the standard water model), yielding up to a 2.68-fold increase in the sampling rate of IDP chain reconfiguration in water and a 16% increase in the lateral diffusion of lipids in a POPC bilayer. Equilibrium properties remained unaffected by the mass scaling, and the speedup was achieved without compromising simulation accuracy. The water model is trivial to implement, has no computational overhead, and should be universally applicable to Martini simulations.

biophysics↗

bioRxiv2026

How the Azadithiolate Ligand Impacts O2-Stability of Group B -Hydrogenase ToHydA

[FeFe]-hydrogenases are metalloenzymes that catalyze the reversible oxidation and production of H2, making them potential candidates for sustainable energy solutions. However, their practical application is restricted by their extreme O2 sensitivity, which leads to irreversible active site degradation. A newly characterized Group B hydrogenase, ToHydA from Thermosediminibacter oceani, has exhibited exceptional O2-stability even after longtime exposure to air. In ToHydA, the highly conserved proton-transporting cysteine (C212) safeguards the H-cluster from O2-induced degradation by formation of the Hinact state. In this study, we investigate the effects of replacing the azadithiolate (ADT) ligand of [2Fe]H with propanedithiolate (PDT), revealing that this substitution prevents the formation of the Hinact and Htrans states observed in ToHydA WT (bearing the ADT ligand). By combining ATR-FTIR spectroscopy and molecular dynamics (MD) simulations, we show that a hydrogen bond between the nitrogen bridgehead of the ADT ligand and the C212 sidechain is crucial for stabilizing these states. The absence of this interaction in ToHydAPDT (bearing the PDT ligand) prevents the C212 sidechain from approaching the Fed center of [2Fe]H, thereby reducing Hinact accumulation. Moreover, as-isolated ToHydAPDT predominantly exhibits the Hhyd state, which is unusual for [FeFe]-hydrogenases with bound PDT ligand. These findings demonstrate how ligand substitution at the [2Fe]H site of ToHydA affects the structural dynamics, offering detailed molecular insights into the ligand-dependent modulation of [FeFe]-hydrogenases.

biophysics↗

bioRxiv2025

Tuning the H2 Production Activity of ToHydA by Molecular Simulation-Informed Protein Engineering

In the context of bio-hydrogen production, the O2-stable Group B [FeFe]-hydrogenase from Thermosediminibacter oceani has attracted significant interest due to its distinctive "TSCCCP" motif near the active site, which contains an additional (third) cysteine residue that is absent in the "TSCCP" motif of standard Group A hydrogenases. The precise role of the additional cysteine residue in H2 production has remained an open question. In this study, we sought to contribute to the understanding of this cysteines role in H2 production by combining molecular dynamics (MD) simulations, site-directed mutagenesis, biochemical assays, and Fourier-transform infrared (FTIR) spectroscopy. Remarkably, a cysteine-to-serine exchange variant (TSSCCP) demonstrated enhanced H2 production activity without compromising the O2-stability of ToHydA, offering new insights into its functional dynamics.

biophysics↗

bioRxiv2025

Predicting the Dynamic Viscosity of High-Concentration Antibody Solutions with a Chemically Specific Coarse-Grained Model

The viscosity of high-concentration protein solutions is a critical parameter in bio-pharmaceutical formulation development. Conventionally, the viscosity is measured and optimized in labor-intensive experimental workflows that require a lot of material. While predicting the viscosity with atomistic molecular dynamics (MD) simulations is feasible, they are computationally prohibitively expensive due to the large system sizes and the long simulation times involved. Coarse-grained MD (CG-MD) simulations significantly reduce computational demands, but evaluating their accuracy and predictive power requires rigorous validation. Here, we assess the capability of the Martini 3 CG force field to predict the viscosity of high-concentration antibody solutions. We show that a refined Martini 3 force field, with optimized protein-protein interactions, can predict the elevated viscosities observed in concentrated solutions of F(ab)2 fragments of the therapeutic monoclonal antibody (mAb) omalizumab. Furthermore, we show that our previously developed Martini 3-exc model for arginine excipients successfully captures the trend of lowering viscosity, as observed in our rheology experiments. These findings open the way to physics-based computational prediction of the properties of dense biopharmaceutical solutions via large-scale MD simulations.

biophysics↗

bioRxiv2025

Robust Estimation of Rotational Diffusion Tensors of Proteins from Molecular Dynamics Simulations

Rotational diffusion is a fundamental physical process that determines the rotational motion of proteins in solution. It plays a role, for example, in molecular association processes and in theories of spectroscopic experiments in solution. In addition to experimental methods, molecular dynamics (MD) simulations have emerged as a powerful method to investigate rotational diffusion. Diffusion models are fitted to rotational correlation functions extracted from the simulations. In this work, we conducted a time-dependent analysis of the model parameters prior to fitting, using extended all-atom MD simulations of ubiquitin as a model system. A comparison to Brownian dynamics (BD) simulations confirms whether the rotational dynamics observed in MD actually follow the theory of Brownian rigid-body diffusion. In addition, the analysis reveals correlation time intervals that are suitable for fitting anisotropic, semi-isotropic, or isotropic diffusion models. To this end, a two-step optimization scheme is employed that combines a global and a local search in parameter space. BD simulations are used to estimate uncertainties of the diffusion coefficients as well as directional uncertainties of the principal axes. We found that ubiquitin exhibits nearly semi-isotropic rotational dynamics, in good agreement with experimental NMR data. The approach is general and can be used to investigate, for example, the rotational diffusion of molecules in various biomolecular environments, or to compute NMR relaxation parameters of proteins. An implementation of the method is freely available at https://github.com/MolSimGroup/rotationaldiffusion.

biophysics↗

bioRxiv2024

Optimized Protein-Excipient Interactions in theMartini 3 Force Field

The high doses of drugs required for biotherapeutics, such as monoclonal antibodies (mAbs), and the small volumes that can be administered to patients by subcutaneous injections pose challenges due to high concentration formulations. The addition of excipients, such as arginine and glutamate, to high concentration protein formulations can increase solubility and reduce the tendency of protein particle formation. Molecular dynamics (MD) simulations can provide microscopic insights into the mode of action of excipients in mAb formulations but require large system sizes and long time scales that are currently beyond reach at the fully atomistic level. Computationally efficient coarse-grained models such as the Martini 3 force field can tackle this challenge but require careful parametrization, testing, and validation. This study extends the popular Martini 3 force field towards realistic protein-excipient interactions of arginine and glutamate excipients, using the Fab domains of the therapeutic mAbs trastuzumab and omalizumab as model systems. A novel all-atom to coarse-grained mapping of the amino acid excipients is introduced, which explicitly captures the zwitterionic character of the backbone. The Fab-excipient interactions of arginine and glutamate are characterized concerning molecular contacts with the Fabs at the single-residue level. The Martini 3 simulations are compared with results from all-atom simulations as a reference. Our findings reveal an overestimation of Fab-excipient contacts with the default interaction parameters of Martini 3, suggesting a too strong attraction between protein residues and excipients. Therefore, we reparametrized the protein-excipient interaction parameters in Martini 3 against all-atom simulations. The excipient interactions obtained with the new Martini 3 mapping and Lennard-Jones (LJ) interaction parameters, coined Martini 3-exc, agree closely with the all-atom reference data. This work presents an improved parameter set for mAb-arginine and mAb-glutamate interactions in the Martini 3 coarse-grained force field, a key step towards large-scale coarse-grained MD simulations of high-concentration mAb formulations and the stabilizing effects of excipients.

biophysics↗

bioRxiv2024

Employing Artificial Neural Networks for Optimal Storage and Facile Sharing of Molecular Dynamics Simulation Trajectories

With the remarkable stride in computing power and advances in Molecular Dynamics (MD) simulation programs, the crucial challenge of storing and sharing large biomolecular simulation datasets has emerged. By leveraging AutoEncoders, a type of artificial neural network, we developed a method to compress MD trajectories into significantly smaller latent spaces. Our method can save up to 98% in disk space compared to xtc, a highly compressed trajectory format from the widely used MD program package GROMACS, thus facilitating storage and sharing of simulation trajectories. Atom coordinates are very accurately reconstructed from compressed data. The method was tested across a diverse sets of biomolecular systems, including folded proteins, intrinsically disordered proteins, phospholipid bilayers, proteinligand complexes, large protein complexes and membrane-bound protein systems. The reconstructed trajectories demonstrated consistent accuracy in recovering key biophysically relevant properties for proteins, lipids and composite systems. The compression efficiency was particularly beneficial for larger systems. This approach enables the scientific community to efficiently store and share large-scale biomolecular simulation data, potentially enhancing collaborative research efforts. The workflow, termed "compresstraj", is implemented in PyTorch and is publicly available at https://github.com/SerpentByte/compresstraj, offering a practical solution for handling the increasing volumes of data generated in biomolecular simulation studies.

biophysics↗

bioRxiv2024

A combined approach to extract rotational dynamics of globular proteins undergoing liquid-liquid phase separation

The formation of protein condensates (droplets) via liquid-liquid phase separation (LLPS) is a commonly observed phenomenon in vitro. Changing the environmental properties with cosolutes, molecular crowders, protein partners, temperature, pressure, etc. was shown to favour or disfavour the formation of protein droplets by fine-tuning the water-water, water-protein and protein-protein interactions. Therefore, these environmental properties and their spatiotemporal fine-tuning are likely to be important also in a cellular context at the existing protein expression levels. One of the key physicochemical properties of biomolecules impacted by molecular crowding is diffusion, which determines the viscoelastic behaviour of the condensates. Here we investigate the change in the rotational diffusion of {gamma}D-crystallin, undergoing LLPS in vitro in aqueous solutions in absence and presence of cosolutes. We studied its rotational dynamics using molecular dynamics simulations (MD), electron paramagnetic resonance (EPR) spectroscopy and fluorescence spectroscopy. MD simulations performed under dilute and crowded conditions show that the rotational diffusion of crystallin in water is retarded by one to two orders of magnitude in the condensed phase. To obtain the rotational dynamics in the dilute phase we used fluorescence anisotropy and to extract the retardation factor in the condensed phase we used spin-labeled {gamma}D-crystallin proteins as EPR viscosity nanoprobes. Aided by a viscosity nanoruler calibrated with solutions at increasing sucrose concentrations, we validate the rotational diffusion retardation predicted by MD simulations. This study underlines the predictive power of MD simulations and showcases the use of a sensitive EPR nanoprobe to extract the viscosity of biomolecular condensates.

biophysics↗

bioRxiv2024

Heterogeneous Slowdown of Dynamics in the Condensate of an Intrinsically Disordered Protein

The high concentration of proteins and other biological macromolecules inside biomolecular condensates leads to dense and confined environments, which can affect the dynamic ensembles and the time-scales of the conformational transitions. Here we use atomistic molecular dynamics (MD) simulations of the intrinsically disordered low complexity domain (LCD) of the human fused in sarcoma (FUS) RNA-binding protein to study how self-crowding inside a condensate affects the dynamic motions of the protein. We found a heterogeneous retardation of the protein dynamics in the condensate with respect to the dilute phase, with large-amplitude motions being strongly slowed by up to two orders of magnitude, whereas small-scale motions, such as local backbone fluctuations and side-chain rotations, are less affected. The results support the notion of a liquid-like character of the condensates and show that different protein motions respond differently to the environment.

biophysics↗

bioRxiv2023

Anionic phospholipids stimulate the proton pumping activity of the plant plasma membrane P-type H+-ATPase

The activity of membrane proteins depends strongly on the surrounding lipid environment. Here, we characterize the lipid stimulation of the plant plasma membrane H+-ATPase AHA2 upon purification and reconstitution into liposomes of defined lipid compositions. We show that the proton pumping activity of AHA2 is stimulated by anionic phospholipids, especially by phosphatidylserine. Molecular dynamics simulations revealed several preferential contact sites for anionic phospholipids in the transmembrane domain of AHA2. These contact sites are partly conserved across functionally different P-type ATPases from several organisms, suggesting a general regulation mechanism by the membrane lipid environment. Our findings highlight the fact that anionic lipids play an important role in the control of H+-ATPase activity.

biochemistry↗

bioRxiv2023

Structural basis for triacylglyceride extraction frommycobacterial inner membrane by MFS transporter Rv1410

Mycobacterium tuberculosis is protected from antibiotic therapy by a multi-layered hydrophobic cell envelope. Major facilitator superfamily (MFS) transporter Rv1410 and the periplasmic lipoprotein LprG are involved in transport of triacylglycerides (TAGs) that seal the mycomembrane. Here, we report a 2.7 [A] structure of a mycobacterial Rv1410 homologue, which adopts an outward-facing conformation and exhibits unusual transmembrane helix 11 and 12 extensions that protrude [~]20 [A] into the periplasm. A small, very hydrophobic cavity suitable for lipid transport is constricted by a unique and functionally important ion-lock likely involved in proton coupling. Combining mutational analyses and MD simulations, we propose that TAGs are extracted from the core of the inner membrane into the central cavity via lateral clefts present in the inward-facing conformation. The periplasmic helix extensions are crucial for lifting TAGs away from the membrane plane and channeling them into the lipid binding pocket of LprG.

molecular biology↗

bioRxiv2022

Integrated assessment of structure and dynamics of solid proteins via accurate solid-state NMR distance information

Understanding macromolecular function, interactions and stability hinges on detailed assessment of conformational ensembles. For solid proteins, accurate elucidation of the spatial aspects of dynamics at physiological temperatures are limited by the qualitative character or low abundance of solid-state NMR internuclear distance information. Here, we demonstrate access to abundant proton-proton internuclear distances for integrated structural biology and chemistry with unprecedented accuracy. Apart from highest-resolution single-state structures, the exact distances enable molecular dynamics (MD) ensemble simulations orchestrated by a dense network of experimental inter-proton distance boundaries gathered in the context of their physical lattices. This direct embedding of experimental ensemble distances into MD will provide access to representative, atomic-level spatial details of conformational dynamics in supramolecular assemblies, crystalline and lipid-embedded proteins, and beyond.

biophysics↗

bioRxiv2022

Epigenetic CpG Duplex Marks Probed by an Evolved DNA Reader via a Well-Tempered Conformational Plasticity

5-methylcytosine (mC) and its TET-oxidized derivatives exist in CpG dyads of mammalian DNA and regulate cell fate, but how their individual combinations in the two strands of a CpG act as distinct regulatory signals is poorly understood. Readers that selectively recognize such novel "CpG duplex marks" could be versatile tools for studying their biological functions, but their design represents an unprecedented selectivity challenge. By mutational studies, NMR relaxation, and MD simulations, we here show that the selectivity of the first designer reader for an oxidized CpG duplex mark hinges on precisely tempered conformational plasticity of the scaffold adopted during directed evolution. Our observations reveal the critical aspect of defined motional features in this novel reader for affinity and specificity in the DNA/protein interaction, providing unexpected prospects for further design progress in this novel area of DNA recognition.

genomics↗

bioRxiv2021

The CTPase activity of ParB acts as a timing mechanism to control the dynamics and function of prokaryotic DNA partition complexes

DNA partitioning CTPases of the ParB family mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromeric region of target DNA molecules and then spread laterally to form large nucleoprotein complexes that serve as docking points for the DNA segregation machinery. Here, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. Moreover, we solve crystal structures of ParB in the pre- and post-hydrolysis state and provide insights into the catalytic mechanism underlying nucleotide hydrolysis. The characterization of CTPase-deficient ParB variants reveals that CTP hydrolysis serves as a timing mechanism to control the sliding time of ParB. Hyperstable clamps are trapped on the DNA, leading to excessing spreading and severe chromosome segregation defects in vivo. These findings clarify the role of the ParB CTPase cycle in partition complex dynamics and function and thus complete our understanding of this prototypic CTP-dependent molecular switch.

microbiology↗

bioRxiv2018

The extracellular gate shapes the energy profile of an ABC exporter

ABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 [A] crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporters conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening resulted in a comparable equilibrium shift and strongly reduced ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state.

biochemistry↗