Biology subjects

Rene Espinosa, J.

Publications and source records attributed to Rene Espinosa, J..

11 recordsLinked to original sources

bioRxiv2026

PepSpace: An Automated, Physics-Driven Directed Evolution Platform for the De Novo Design of Specific Peptide Binders and Condensate Modulators

Intrinsically disordered proteins, topologically complex protein surfaces, and biomolecular condensates regulate essential cellular processes but remain difficult to drug with conventional small molecules. Here, we present PepSpace, a physics-driven directed-evolution platform coupling residue-resolution coarse-grained molecular dynamics with a multi-objective genetic algorithm. PepSpace designs peptide binders by rewarding target engagement while penalizing peptide self-association and off-target binding. Evaluated alongside deep-learning generative models across multiple targets, PepSpace-designed peptides achieve predicted interaction strengths up to two orders of magnitude greater than machine-learning designs, while reproducing experimentally observed binding hierarchies and revealing distinct binding modes. For PIEZO1, PepSpace designs 25-mer peptides that preferentially engage a defined 30-residue intracellular beam epitope while suppressing interactions with the remainder 2,547-residues of the protein. For CHERP biomolecular condensates, dual-action peptides weaken native intermolecular contacts and destabilize the condensed phase. PepSpace provides a physically grounded framework for de novo peptide design against dynamic protein targets and biomolecular condensates, with explicit control over specificity, solubility, and collective interactions.

biophysics↗

bioRxiv2026

Length Scale-Dependent Dynamics in Electrostatic Protein Coacervates

Biomolecular condensates formed by complex coacervation of highly charged proteins provide a powerful framework to understand how microscopic interactions give rise to macroscopic material properties. Atomistic molecular dynamics simulations provide detailed insights but remain limited in accesing the spatio-temporal scales relevant for condensate behavior. Here, we use the residue-level coarse-grained Mpipi-Recharged model to investigate condensates formed by ProT and positively charged partners, including histone H1, protamine, poly-lysine, and poly-arginine. Material properties, in this context, provide a stringent experimental benchamark for coarse-grained models. Our model reproduces salt-dependent phase behavior, protein binding affinities, and sequence-specific stability trends in agreement with in vitro experiments, despite the fact that material properties were not included in the model parametrization. We then establish a direct link between protein dynamics and macroscopic material properties by quantifying monomeric diffusion, conformational reconfiguration, and translational mobility within the dense phase, and relating these to condensate viscosity. By comparing dynamics across dense and dilute phases, we uncover a pronounced length scale-dependent behavior. While residue-level binding and unbinding events remain equally fast in both phases, protein reconfiguration time and self-diffusion are significantly slowed down within the condensates. This decoupling reveals how fast intermolecular interactions coexist with slow mesoscale condensate dynamics depending on the molecular length scale. Together, our results establish a predictive framework that links encoded sequence intermolecular forces and multiscale dynamics to the emergent material properties of complex biomolecular condensates.

biophysics↗

bioRxiv2026

Computational mapping of antibody-receptor energy landscapes to predict membrane internalization

Antibody internalization is critical for the action of antibody-drug conjugates, yet antibody discovery pipelines typically prioritize binding affinity rather than functional internalization. Here we show that molecular dynamics simulations can map the binding energy landscape between antibody clones and the membrane protein JAM-A, enabling computational predictions of antibody internalization. Using the sequences of newly generated anti-JAM-A monoclonal antibodies (mAbs), we perform atomistic potential-of-mean force simulations to evaluate the binding free energy to the JAM-A receptor and their interaction fingerprint at residue-level resolution. We find that internalizing mAb derived from different hybridomas exhibit a unique membrane-oriented contact topology that promotes cooperative receptor-receptor interactions, lowering the energetic barrier for early endocytic events. Reconstruction of the receptor binding energy landscape further reveals that electrostatic interactions between charged residues and multivalent cation-{pi} and polar interactions correlate with successful mAb internalization in ovarian cancer cells. In contrast, strong binding affinity of the fragment antigen-binding domain correlates with poor internalization. Together, our results establish molecular dynamics-guided clonal selection as a predictive framework for optimizing internalizing therapeutic antibodies and provide mechanistic insight into how antibody binding reshapes membrane-proximal receptor energetics to drive endocytosis.

biophysics↗

bioRxiv2026

Interplay between Local Diffusion, Concentration, and Inter-Protein Alignment Promotes Cross-β-Sheet Transitions at Condensate Interfaces

The interface separating a biomolecular condensate from its surroundings is increasingly recognised as a unique physical and chemical environment that actively regulates condensate behaviour. It controls surface tension, molecular organisation and chemical reactivity, and is also where pathological ageing often begins, with several proteins nucleating cross-{beta}-sheet fibrils preferentially at condensate surfaces. But does ageing emerge there because of protein-specific chemistry, or a more general physical mechanism? Using coarse-grained molecular dynamics simulations, we show that cross-{beta}-sheet nucleation generically occurs preferentially at condensate interfaces across systems of increasing chemical complexity, from simple homopolymers to patterned amphiphilic sequences. In all systems studied, interface-driven ageing has a common physical origin and can be further modulated, but not driven, by protein sequence. Specifically, terminal domains at the condensate interface gain mobility while remaining densely packed and mutually aligned, creating favourable conditions for {beta}-sheet nucleation. Ageing is amplified in amphiphilic sequences, where hydrophilic regions act as surfactants facing the dilute phase, while aggregation-prone hydrophobic domains cluster just beneath the surface, concentrating the nucleation there. This surfactant-like organisation lowers the interfacial free energy, which falls systematically as sequence asymmetry grows. By linking molecular interactions to mesoscale behaviour, these results cast the interface as a central regulator of ageing and reveal a sequence-independent physical mechanism for the liquid-to-solid transition underlying pathological aggregation in biomolecular condensates.

biophysics↗

bioRxiv2025

Multi-scale structure of chromatin condensates rationalizes phase separation and material properties

Biomolecular condensates, compartments that concentrate molecules without surrounding membranes, are integral to numerous cellular processes. The structure and interaction networks of molecules within condensates remain poorly understood. Using cryo-electron tomography and molecular dynamics simulations we elucidated the structure of phase separated chromatin condensates across scales, from individual amino acids to network architecture. We found that internucleosomal DNA linker length controls nucleosome arrangement and histone tail interactions, shaping the structure of individual chromatin molecules both within and outside condensates. This structural modulation determines the balance between intra- and intermolecular interactions, which in turn governs the molecular network, thermodynamic stability, and material properties of chromatin condensates. Mammalian nuclei contain dense clusters of nucleosomes whose non-random organization is mirrored by the reconstituted condensates. Our work explains how the structure of individual chromatin molecules ultimately determines physical properties of chromatin condensates, with implications for cellular chromatin organization.

biophysics↗

bioRxiv2024

Nucleosome Spacing Can Fine-Tune Higher Order Chromatin Assembly

Cellular chromatin displays heterogeneous structure and dynamics, properties that control diverse nuclear processes. Models invoke phase separation of conformational ensembles of chromatin fibers as a mechanism regulating chromatin organization in vivo. Here we combine biochemistry and molecular dynamics simulations to examine, at single base-pair resolution, how nucleosome spacing controls chromatin phase separation. We show that as DNA linkers extend from 25 bp to 30 bp, as examplars of 10N+5 and 10N (integer N) bp lengths, chromatin condensates become less thermodynamically stable and nucleosome mobility increases. Simulations reveal that this is due to trade-offs between inter- and intramolecular nucleosome stacking, favored by rigid 10N+5 and 10N bp linkers, respectively. A remodeler can induce or inhibit phase separation by moving nucleosomes, changing the balance between intra- and intermolecular stacking. The intrinsic phase separation capacity of chromatin enables fine tuning of compaction and dynamics, likely contributing to heterogeneous chromatin organization in vivo.

biophysics↗

bioRxiv2024

Charged peptides enriched in aromatic residues decelerate condensate ageing driven by cross-beta-sheet formation

Biomolecular condensates, formed through liquid-liquid phase separation, play wide-ranging roles in cellular compartmentalization and biological processes. However, their transition from a functional liquid-like phase into a solid-like state--usually termed as condensate ageing--represents a hallmark associated with the onset of multiple neurodegenerative diseases. In this study, we design a computational pipeline to explore potential candidates, in the form of small peptides, to regulate ageing kinetics in biomolecular condensates. By combining equilibrium and non-equilibrium simulations of a sequence-dependent residue-resolution force field, we investigate the impact of peptide insertion--with different composition, patterning, and net charge--in the condensate phase diagram and ageing kinetics of archetypal proteins driving condensate ageing: TDP-43 and FUS. We reveal that small peptides composed of a specific balance of aromatic and charged residues can substantially decelerate ageing over an order of magnitude. The mechanism is controlled through condensate density reduction induced by peptide self-repulsive electrostatic interactions that specifically target protein regions prone to form cross-{beta}-sheet fibrils. Our work proposes an efficient computational framework to rapidly scan the impact of small molecule insertion in condensate ageing and develop novel pathways for controlling phase transitions relevant to disease prevention.

biophysics↗

bioRxiv2024

Dynamically arrested condensate fusion creates complex structures with varying material properties

The cell nucleus and cytosol contain numerous biomolecular condensates which dynamically reshape, fuse and split to accomplish precise compartmentalization of the cell material. While it has been observed that some condensates rapidly coalesce, some others only attach to each other, or do not establish persistent interactions over time. Here, we explain these observations through optical tweezers and Molecular Dynamics simulations focusing on two condensate-forming, RNA-binding proteins--FUS and G3BP1--strongly involved in RNA metabolism and stress responses. We find that the fusion of pure droplets formed by these proteins can give rise to multiphase single-component condensates exhibiting notably different densities, architectures, and material properties. Such behaviour is dictated by the relative timescales of condensate fusion and protein internal mixing. A critical parameter controlling this interplay is the extent of ageing that condensates display; e.g., their progressive hardening driven by the accumulation of inter-protein {beta}-sheet assemblies over time. Strikingly, different degrees of ageing in fusing droplets can lead single-component condensates to form diverse architectures including concentric drops or two-sided condensates. Overall, our results highlight a mechanism, based on the temporal coupling between ageing, fusion, and mixing rate, by which biomolecular condensates form multiphasic structures with markedly different material properties, and hence potentially distinct biological roles.

biophysics↗

bioRxiv2024

Phase behaviour of hnRNPA1 low-complexity domain mutants described by different sequence-dependent models

Intracellular liquid-liquid phase separation (LLPS) of proteins and nucleic acids is a fundamental mechanism by which cells compartmentalize their components and perform essential biological functions. Molecular simulations play a crucial role in providing microscopic insights into the physicochemical processes driving this phenomenon. In this study, we systematically compare six state-of-the-art sequence-dependent, residue-resolution models to evaluate their performance in reproducing the phase behaviour and material properties of condensates formed by seven variants of the low-complexity domain (LCD) of the hnRNPA1 protein (A1-LCD)--a protein implicated in the pathological liquid-to-solid transition of stress granules. Specifically, we assess the HPS, HPS-cation-{pi}, HPS-Urry, CALVADOS2, Mpipi, and Mpipi-Recharged models in their predictions of the condensate saturation concentration, critical solution temperature, and condensate viscosity for the A1-LCD variants. Our analyses demonstrate that, among the tested models, Mpipi, Mpipi-Recharged, and CALVADOS2 provide accurate descriptions of the critical solution temperatures and saturation concentrations for the various A1-LCD variants tested. Regarding the prediction of material properties for condensates of A1-LCD and its variants, Mpipi-Recharged stands out as the most reliable model. Overall, this study benchmarks a range of residue-resolution coarse-grained models for the study of the thermodynamic stability and material properties of condensates and establishes a direct link between their performance and the ranking of intermolecular interactions these models consider.

biophysics↗

bioRxiv2023

Mechano-dependent sorbitol accumulation supports biomolecular condensate

Biomolecular condensates regulate a wide range of cellular functions from signaling to RNA metabolism1, 2, yet, the physiologic conditions regulating their formation remain largely unexplored. Biomolecular condensate assembly is tightly regulated by the intracellular environment. Changes in the chemical or physical conditions inside cells can stimulate or inhibit condensate formation3-5. However, whether and how the external environment of cells can also regulate biomolecular condensation remain poorly understood. Increasing our understanding of these mechanisms is paramount as failure to control condensate formation and dynamics can lead to many diseases6, 7. Here, we provide evidence that matrix stiffening promotes biomolecular condensation in vivo. We demonstrate that the extracellular matrix links mechanical cues with the control of glucose metabolism to sorbitol. In turn, sorbitol acts as a natural crowding agent to promote biomolecular condensation. Using in silico simulations and in vitro assays, we establish that variations in the physiological range of sorbitol, but not glucose, concentrations, are sufficient to regulate biomolecular condensates. Accordingly, pharmacologic and genetic manipulation of intracellular sorbitol concentration modulates biomolecular condensates in breast cancer - a mechano-dependent disease. We propose that sorbitol is a mechanosensitive metabolite enabling protein condensation to control mechano-regulated cellular functions. Altogether, we uncover molecular driving forces underlying protein phase transition and provide critical insights to understand the biological function and dysfunction of protein phase separation.

cell biology↗

bioRxiv2022

Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

PRC1 (Polycomb repressive complex 1) plays a significant role in cellular differentiation and development by repressing lineage-inappropriate genes. PRC1 proteins phase separate to form Polycomb condensates (bodies) that are multi-component hubs for silencing Polycomb target genes; however, the molecular principles that underpin the condensate assembly and biophysical properties remain unknown. Here, by using biochemical reconstitution, cellular imaging, and multiscale molecular simulations, we show that PRC1 condensates are assembled via a scaffold-client liquid-liquid phase separation (LLPS) model by which Chromobox 2 (CBX2) is the scaffold and other subunits of the CBX2-PRC1 complex act as clients. The clients induce a reentrant phase transition of CBX2 condensates in a concentration-dependent manner. The composition of the multi-component, heterotypic LLPS systems directs the assembly and biophysical properties of CBX2-PRC1 condensates and selectively promotes the formation of CBX4-PRC1 condensates, but specifically dissolves condensates of CBX6-, CBX7-, and CBX8-PRC1. Additionally, the composition of CBX2-PRC1 condensates controls the enrichment of CBX4-, CBX7-, and CBX8-PRC1 into condensates but the exclusion of CBX6-PRC1 from condensates. Our results show the composition- and stoichiometry-dependent scaffold-client assembly of multi-component PRC1 condensates and supply a conceptual framework underlying the molecular basis and dynamics of Polycomb condensate assembly.

biochemistry↗