The quantitative prediction of biomolecular recognition is crucial to molecular science. The challenge is not merely structural determination but the prediction of (thermo)dynamic and kinetic observab...

Multimicrosecond MD simulations reveal that dual phosphorylation at Ser226/Ser255 of Hsp90β acts as a molecular clamp, rigidifying the overall structure, propagating allosteric coordination changes to...

The molecular chaperone HSP90 drives the folding and activation of a broad spectrum of client proteins through dynamic, transient multiprotein assembl…

Molecular dynamics simulations provide essential atomistic insights into the organization and dynamics of complex biological membranes. However, the equilibration of large-scale, curved lipid assemblies at the all-atom level remains a significant challenge. Standard construction approaches, such as wrapping planar bilayers onto spherical meshes, frequently reverberate into structural instabilities, including membrane holes, infiltrated water, lipid flipping, and nonequilibrated densities, which hinder stable production simulations. Here, we present ReVesicle, an iterative equilibration protocol designed to restore and stabilize quasi-spherical lipid vesicles with complex compositions and large dimensions. The proposed protocol combines selective identification and removal of infiltrated water molecules and flipped lipids with short nonequilibrium MD cycles and anisotropic pressure equilibration. These steps are organized into a modular, iterative sequence that progressively recovers bilayer continuity while preserving the vesicle geometry and enabling global density relaxation. Local vacuum-induced stress generated during nonequilibrium phases promotes lipid tail melting and hole curation, while anisotropic equilibration allows relaxation of box dimensions and system density. To demonstrate the robustness of ReVesicle, we applied the protocol to six biologically realistic vesicle systems: synaptic vesicles, plasma membranes, late endosomes, exosomes, mitochondria-derived vesicles, and the HIV-1 lipid envelope. These systems span diameters from 40 to 105 nm and reach total sizes of up to ∼150 million atoms with heterogeneous and asymmetric lipid compositions. Across all cases, ReVesicle consistently converges to continuous, tightly packed bilayers. Structural and biophysical analyses, including vesicle diameter, sphericity, area per lipid, and lipid acyl-chain order parameters, indicate preservation of quasi-spherical geometry and structural integrity. Overall, ReVesicle provides a reproducible framework for equilibrating large, heterogeneous lipid vesicles suitable for downstream all-atom simulations of complex biological environments.

The dissociation of a ligand bound to a receptor is a rare event occurring on a timescale that is far longer than can be afforded by standard simulation methodo

Oligosaccharyltransferase (OST) catalyses the key step of N-glycosylation, transferring immature N-glycans to select Asn residues in nascent proteins in the endoplasmic reticulum (ER). Asn are more li...

Machine learning (ML) and deep learning (DL) methodologies have significantly advanced drug discovery and design in several aspects. Additionally, the integration of structure-based data has proven to...

Essential chaperones heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) collaborate in oncoprotein folding. Dual inhibition of these chap…

Post-translational modification (PTM) of proteins regulates cellular proteostasis by expanding protein functional diversity. This naturally leads to increased proteome complexity as the result of PTM ...

Antimicrobial resistance (AMR) is a global threat, with Burkholderia species contributing significantly to difficult-to-treat infections. The Pen family of β-lactamases are produced by all Burkholderi...

Screen of minimalistic enhancers in blood progenitor cells demonstrates widespread dual activator-repressor function of transcription factors (TFs) and enables the model-guided design of cell-state-sp...

Phosphodiester groups occur ubiquitously in nature, e.g. in nucleic acids or in cyclic (di-)nucleotides important for signal transduction. Proteins often use polar or positively charged amino acids t...

Nature Communications - Mutations cause impairment of function in the chaperone Hsp60. Here, the authors investigate their impact with MD simulations from the monomer to the 28-mer complex, and...

Accelerated SuFEx Click Chemistry (ASCC) is a powerful method for coupling aryl and alkyl alcohols with SuFEx-compatible functional groups. With its hallmark favorable kinetics and exceptional product...

Mutations cause impairment of function in the chaperone Hsp60. Here, the authors investigate their impact with MD simulations from the monomer to the 28-mer complex, and show the pervasive effects of ...

Enzymes are the quintessential green catalysts, but realizing their full potential for biotechnology typically requires improvement of their biomolecular properties. Catalysis enhancement, however, is often accompanied by impaired stability. Here, we show how the interplay between activity and stability in enzyme optimization can be efficiently addressed by coupling two recently proposed methodologies for guiding directed evolution. We first identify catalytic hotspots from chemical shift perturbations induced by transition-state-analogue binding and then use computational/phylogenetic design (FuncLib) to predict stabilizing combinations of mutations at sets of such hotspots. We test this approach on a previously designed de novo Kemp eliminase, which is already highly optimized in terms of both activity and stability. Most tested variants displayed substantially increased denaturation temperatures and purification yields. Notably, our most efficient engineered variant shows a ∼3-fold enhancement in activity (kcat ∼ 1700 s–1, kcat/KM ∼ 4.3 × 105 M–1 s–1) from an already heavily optimized starting variant, resulting in the most proficient proton-abstraction Kemp eliminase designed to date, with a catalytic efficiency on a par with naturally occurring enzymes. Molecular simulations pinpoint the origin of this catalytic enhancement as being due to the progressive elimination of a catalytically inefficient substrate conformation that is present in the original design. Remarkably, interaction network analysis identifies a significant fraction of catalytic hotspots, thus providing a computational tool which we show to be useful even for natural-enzyme engineering. Overall, our work showcases the power of dynamically guided enzyme engineering as a design principle for obtaining novel biocatalysts with tailored physicochemical properties, toward even anthropogenic reactions.

Optical tweezers are widely used in the study of biological macromolecules but are limited by their one-directional probing capability, potentially missing critical conformational changes. Combining ...

Cell Death & Disease - Point mutations of the mitochondrial chaperone TRAP1 affect its functions and pro-neoplastic activity

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The existence of the phenomenon of enhanced enzyme diffusion (EED) has been a topic of debate in recent literature. One proposed mechanism to explain the origin of EED is oligomeric enzyme dissociation. We used mass photometry (MP), a label-free single-molecule technique, to investigate the dependence of the oligomeric states of several enzymes on their ligands. The studied enzymes of interest are catalase, aldolase, alkaline phosphatase, and vanillyl-alcohol oxidase (VAO). We compared the ratios of oligomeric states in the presence and absence of the substrate as well as different substrate and inhibitor concentrations. Catalase and aldolase were found to dissociate into smaller oligomers in the presence of their substrates, independently of inhibition, while for alkaline phosphatase and VAO, different behaviors were observed. Thus, we have identified a possible mechanism which explains the previously observed diffusion enhancement in vitro. This enhancement may occur due to the dissociation of oligomers through ligand binding.

Often dismissed as the poor cousin of the sciences, chemistry has revealed natural laws that illuminate our Universe

The assembly of proteins in membranes plays a key role in many crucial cellular pathways. Despite their importance, characterizing transmembrane assembly remains challenging for experiments and simula...

"How to evaluate de novo designs proposed by a generative model?" Despite the transformative potential of generative deep learning in drug discovery, this seemingly simple question has no clear answer...

An orthogonal alphaviral RNA replication system with chemically inducible control of RNA mutagenesis enables RNA-based directed evolution in mammalian cells.

Functional systematic pseudotype virus-based analysis of the ability of diverse animal viruses to enter into a panel of well-characterized human cell lines shows a broad compatibility between animal v...

Systematic comparison of SARS-CoV-2 spike proteins from pre- and post-Omicron variants in cell lines, primary respiratory epithelial cells and Syrian hamsters show distinct phenotypic trajectories in ...