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A Fine-tuned ProtGPT2 (transformer model) for Predicting more Virulent SARS-CoV-2 variants and understanding its virulence by biophysical methods.

The emergence of Variants of Concern (VOCs) of SARS-COV2 with increased virulence and transmissibility has been linked to multiple mutations in the RBD region, altering their antigenic properties. In this study, we used a specialized ProtGPT2 model trained on the SARS-COV2 spike protein to forecast probable mutations on the spike protein that have not yet emerged. Upon prediction, we systematically studied the stability of single-site and multisite mutations using unbiased molecular dynamics simulations. Binding free energies were used to study the physicochemical significance of the mutations and their affinity to human ACE2 receptor. Our results show that there are specific hot-spots that mutate in the spike protein that enhance binding affinity through electrostatic and improved non-bonded interactions and highlight the role of specific energetic contributions in viral adaptation and infectivity. Our analysis revealed that the reduction of a disulphide bridge within sites 480-488 lowered the binding free energy and increased the flexibility of the loop region, enhancing its interface interaction with ACE, leading to a more virulent variant than Omicron.

biophysics↗

Exploration of the influence of environmental changes on the conformational and amyloidogenic landscapes of the zinc finger protein DPF3a by combining biophysical and molecular dynamics approaches

In the past few years, the double PHD fingers 3 (DPF3) protein isoforms (DPF3b and DPF3a) have been identified as new amyloidogenic intrinsically disordered proteins (IDPs). Although such discovery is coherent and promising in the light of their involvement in proteinopathies, their amyloidogenic pathway remains largely unexplored. As environmental variations in pH and ionic strength are relevant to DPF3 pathophysiological landscape, we therefore enquired the effect of these physicochemical parameters on the protein structural and prone-to-aggregation properties, by focusing on the more disordered DPF3a isoform. In the present study, we exploited in vitro and in silico strategies by combining spectroscopy, microscopy, and all-atom molecular dynamics methods. Very good consistency and complementary information were found between the experiments and the simulations. Acidification unequivocally abrogated DPF3a fibrillation upon maintaining the protein in highly hydrated and expanded conformers due to extensive repulsion between positively charged regions. In contrast, alkaline pH delayed the aggregation process due to loss in intramolecular contacts and chain decompaction, the extent of which was partly reduced thanks to the compensation of negative charge by arginine side chains. Through screening attractive electrostatic interactions, high ionic strength conditions (300 and 500 mM NaCl) shifted the conformational ensemble towards more swollen, heterogeneous, and less H-bonded structures, which were responsible for slowing down the conversion into {beta}-sheeted species and restricting the fibril elongation. For defining the self-assembly pathway of DPF3a, we unveiled that the protein amyloidogenicity intimately communicates with its conformational landscape, which is particularly sensitive to modification of its physicochemical environment. As such, understanding how to modulate DPF3a conformational ensemble will help designing novel protein-specific strategies for targeting neurodegeneration. HighlightsO_LIDPF3a is a polyampholyte IDP, structurally sensitive to environmental changes. C_LIO_LIDPF3a amyloid pathway and propensity can be modulated by pH and ionic strength. C_LIO_LIAcidic condition inhibits fibrillation and maintains DPF3a in an extended state. C_LIO_LIAlkaline pH and ionic strength delay fibrillation by reducing structure collapse. C_LIO_LIDPF3a fibrils exhibit condition-dependent optical-morphological properties. C_LI

biophysics↗

Integrating computational and experimental biophysics reveals novel insights into the RAD51-BRC4 interaction

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/642044v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@32e13org.highwire.dtl.DTLVardef@c60a6forg.highwire.dtl.DTLVardef@1d6fdceorg.highwire.dtl.DTLVardef@1b10541_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG The interaction between the RAD51 and BRCA2 proteins is central for homologous recombination, a crucial pathway ensuring high-fidelity DNA repair. Recruitment of RAD51 involves eight highly conserved regions on BRCA2, named BRC repeats. To date, only the interaction between the fourth BRC repeat (BRC4) and the RAD51 C-terminal domain has been structurally characterized, while the complex of full-length RAD51 with the peptide still remains elusive. Here, we report an integrative experimental and in silico approach to reconstruct the conformational ensemble in solution for full length RAD51 in complex with BRC4. We combined AlphaFold2, crosslinking mass spectrometry (XL-MS) and small angle x-ray scattering (SAXS) data with molecular dynamics simulations (MD). These data show that the full-length RAD51-BRC4 complex is a mixture of compact and elongated conformations. Detailed analysis of the reweighted ensemble, achieved through the maximum entropy principle, identifies key residues at the N-terminal-BRC4 interface mediating complex conformational dynamics. Our evidence provides robust atomic-level insights into the interaction of RAD51 and BRC4. These findings are crucial for understanding the molecular features underlying the recognition between RAD51 and BRCA2, which are essential for developing therapeutic intervention strategies in cancer treatment.

biophysics↗

Structural, biophysical and biological analysis and characterisation of IRF4 DNA-binding domain mutations associated with multiple myeloma

IRF4, a transcription factor in the interferon regulatory factor family, is a key regulator in immune cell differentiation indicated to have an essential role in the development of lymphoid malignancies. Genome-wide association studies previously identified a set of overlapping mutations within the IRF4 DNA-binding domain in T-cell lymphoma and multiple myeloma, several of which appeared to be associated with better prognosis. Mapping these mutations to the known crystal structure of the IRF4:PU.1:DNA ternary complex and a new structure of the IRF4 DNA-binding domain in the apo state suggested they might interfere with DNA-binding, directly or via destabilisation of domain structure. We characterised these cancer-associated IRF4 mutants experimentally using the recombinant IRF4 DNA-binding domain (DBD) in vitro and examined the clinically relevant mutant K123R in cellulo. Using fluorescence polarisation, surface plasmon resonance, differential scanning fluorimetry and molecular dynamics, we find that mutation may give rise to significant differences in DNA-binding kinetics and thermal stability without compromising the affinity of IRF4 DNA-binding. The K123R IRF4 mutant showed increased transcriptional activity via a luciferase reporter assay and increased nuclear partitioning, which may be preferentially selected for in multiple myeloma. We discuss our observations in relation to the improved prognosis conferred by this mutation.

biophysics↗

High-Level Expression, Purification and Biophysical Characterization of GPI-anchored native-like human Prion Protein using Leishmania tarentolae

The human prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-linked membrane-bound glycoprotein, containing two glycosylation sites. Human PrP is associated with a number of neurodegenerative diseases, called transmissible spongiform encephalopathies (TSE). Pathogenesis involves a structural conversion of the cellular form (PrPC), rich in -helical and random coil structure, into the scrapie form (PrPSc) characterized by parallel in register intermolecular {beta}-sheet conformation. To get a better understanding of this structural conversion, it is crucial to first characterize the non-pathogenic cellular isoform including all posttranslational modifications, like GPI-anchoring and native-like human glycosylation pattern. So far, studies on PrPC or PrPSc as well as the transition from one state to the other rely on non-native constructs of PrP studied far away from physiological conditions. We, therefore, established the expression of GPI-linked human PrP with close to native glycosylation pattern (native-like human PrP) using the eukaryotic LEXSY expression system in Leishmania tarentolae. This expression system has the added advantage that it allows for large-scale production of the native-like human PrP, which results in [~]1 mg purified protein per liter culture. Sedimentation velocity analysis and far-UV circular dichroism spectroscopy confirm the high structural homogeneity and monomeric native-like conformation of the purified GPI-anchored human PrP.

biophysics↗

A high-throughput platform for biophysical antibody developability assessment to enable AI/ML model training

Antibodies must bind their targets with high affinity and specificity to achieve useful therapeutic activity. They must also possess suitable developability properties (e.g., thermostability, solubility, viscosity, polyreactivity) to ensure favorable manufacturing, formulation, and in vivo performance. Both binding and developability properties are inherent to a given antibody amino acid sequence. Identification or selection of antibodies possessing suitable binding characteristics is now routine, and de novo computational design models, trained on extensive complementarity-determining region sequence and structural data, are rapidly improving. Developability properties, however, remain difficult to predict largely due to insufficient training data, with empirical testing being heavily used to avoid challenges in late-stage antibody development. To fill this gap, we built a high-throughput antibody developability assay platform designed to generate the large datasets needed to train improved machine learning (ML) models. We optimized and automated known developability assays [Jain et al., 2017], and developed a robust integrated data analytics pipeline. Here we report data on 246 antibodies--representing 106 approved, 135 clinical-stage, and 5 preregistration/withdrawn molecules--across a panel of 10 developability assays, in a "tidy data" format suitable for AI/ML modeling. We used these data to develop an XGBoost [Chen et al., 2016] ML model that better predicts similarity to approved antibodies compared to conventional use of developability warning thresholds. Additionally, we confirm that preliminary predictive models do improve with more training data. Our high-throughput PROPHET-Ab platform enables data generation at the scale needed to develop improved ML models to predict antibody developability. SignificanceSuccessful antibody drugs exhibit important "developability" properties, beyond tight and specific binding to their target, including high expressibility, high stability and solubility, low aggregation propensity, low viscosity, low polyreactivity, and long in vivo half-life. Collectively, developability properties predict favorable manufacturing, storage, administration, and safety, and deficiencies in these properties increase risk for clinical failure. Despite progress in developing machine learning models to predict structure and binding, antibody developability models lag, largely due to a lack of sufficiently large training datasets. We have built a high-throughput platform, PROPHET-Ab, that enables data generation at the scale needed to train improved AI/ML models to predict antibody developability.

biophysics↗

Biophysics of the Coronavirus-Membrane Interaction: Role of Nonequilibrium Binding Energy

Scientists seemed not to have explored mechanical kinetic energy, a path function for the determination of nonequilibrium binding energy (NEBE). The study explored mechanical kinetic energy, a path function in particles that requires nonequilibrium binding energy (NEBE) to counteract it. It aimed to show that there was a minimally sufficient NEBE to counteract mechanical kinetic energy, using literature data to evaluate derived equations and computations. As is typical, diffusivities increase with temperature; however, near binding, the binding limitations cause the diffusivities (1.519 [->] 2.784 exp. (- 15) m2/s for D variant; 1.415[->]2.577 exp. (- 15) m2/s for G variant) to be lower than when they are far from binding (4.36 [->]7.99 exp. (- 15) m2/s for D variant; 4.151[->]7.611 exp. (- 15) m2/s for G variant). With breath emission equal to 1.29 exp. (7)/m3, the NEBEs were 656.020 and 663.212 kcal/mol for Delta and Omicron variants of SARS-CoV-2, respectively; and with 27.9 exp. (7)/m3, the corresponding values were 568.921 and 633 kcal/mol; with a breath emission rate equal to 9.31 exp. (+6)/hr., the NEBEs for the Omicron variant in 1 hr. and in 1 min. were 651.703 and 683.353 kcal/mol, respectively; with 201 exp. (+6)/hr., the corresponding values were 444.157 and 663.212 kcal/mol. The G variant of SARS-CoV-2 showed higher NEBEs (952[->]671 kcal/mol) than D variants (834[->]588 kcal/mol), corresponding to 293.15[->]318.15 K. The decreasing trend in maximum NEBE [Formula] with rising temperature implies that the binding affinity of the virus may be attenuated at higher temperatures. It is, therefore, medically plausible to administer airborne, parenteral, oral, etc., drugs at temperatures above body temperatures that are tolerable and in a controlled fashion. Future studies should be directed to a definite determination of the size and molar mass of variants of SARS-CoV-2.

biophysics↗

Three biophysical constraints determine the variation of structural divergence among residues in enzyme evolution

AO_SCPLOWBSTRACTC_SCPLOWIn enzyme evolution, structural divergence varies among residues, forming residue-dependent structural divergence profiles. The evolutionary constraints that determine these profiles remain unclear. We build on the Mutation-Stability-Activity (MSA) model, a mechanistic mutation-selection model previously developed for sequence evolution. In the MSA model, mutations become fixed or are lost depending on their effects on stability and activity, with parameters aS and aA controlling selection on stability and activity, respectively. The Linearly Forced Elastic Network Model (LFENM) is used to calculate mutational effects on structure, stability, and activity. As substitutions accumulate, structural changes build up unevenly across residues, producing a structural divergence profile that depends on how each residue responds to mutation and on how strongly selection acts on stability and activity. Applied to 34 enzyme families, the MSA model recapitulates observed structural divergence profiles, and nested model comparisons show that mutation, stability, and activity constraints each contribute. However, the balance among these constraints varies widely across families: mutation always contributes substantially, but stability and activity contributions range from negligible to dominant, so any of the three can prevail in a given family. These variations have distinct origins: the mutation contribution depends on how unevenly the proteins flexibility is distributed across residues, while the stability and activity contributions depend on how strongly selection acts, as quantified by aS and aA. The MSA model thus recovers family-specific selection strengths from structural divergence profiles, suggesting these profiles encode information not only about enzyme architecture but also about the selective regime under which enzymes evolve.

biophysics↗

Affinity Selection-Mass Spectrometry Coupled with Biophysical Validation Enables Proof-of-Concept Discovery of CHI3L1 Binders

Chitinase-3-like protein 1 (CHI3L1) is a multifunctional extracellular glycoprotein implicated in tumor progression, immune suppression, and fibrosis, making it an attractive but challenging therapeutic target. To explore its chemical tractability, we applied an affinity selection-mass spectrometry (AS-MS) workflow to screen 10,000 small molecules for CHI3L1 binding. The screen yielded 124 initial hits with a hit rate of 1.24%, which were prioritized based on chemical suitability, and six candidates were advanced for validation using microscale thermophoresis (MST). Among these, compound A9 exhibited a clear, dose-dependent binding response in MST with a Kd of 182 {+/-} 18 {micro}M. Molecular docking supported these findings, revealing that A9 forms hydrophobic and hydrogen-bonding interactions within a defined pocket of the CHI3L1 structure. Although modest in affinity, A9 represents the first small molecule binder of CHI3L1 identified through AS-MS. This study provides a proof-of-concept demonstration that CHI3L1 can be chemically engaged using AS-MS, establishing a foundation for future medicinal chemistry optimization and the development of chemical probes targeting this previously undruggable extracellular protein. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/681114v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@12959c2org.highwire.dtl.DTLVardef@1c3e3a4org.highwire.dtl.DTLVardef@1962a04org.highwire.dtl.DTLVardef@1a0a2c4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

Computational Design and Biophysical Validation of Macrocyclic Peptides as Inhibitors of SLIT2/ROBO1 interaction

The SLIT2/ROBO1 signaling axis regulates cellular migration and angiogenesis but also contributes to tumor progression and immune evasion in glioblastoma. Targeting this pathway with small molecules or antibodies remains challenging due to the shallow and extended nature of the SLIT2/ROBO1 interface. Here, we report the first computational design and experimental validation of macrocyclic peptides that inhibit SLIT2/ROBO1 binding. Twenty peptides were generated through a structure-guided interface mapping approach (Des3PI 2.0) and ranked using a contact-based scoring function. The top candidates were synthesized and evaluated using time-resolved fluorescence resonance energy transfer (TR-FRET) and biolayer interferometry (BLI) assays. Among the SLIT2-targeting peptides, SP4 and SP3 showed the most pronounced inhibition in TR-FRET and BLI, confirming direct binding to the SLIT2/ROBO1 interface. The lead peptide SP4 also demonstrated favorable in vitro pharmacokinetic properties, including strong stability in simulated intestinal fluid, high plasma integrity, and moderate metabolic stability in rat liver microsomes. Collectively, this work establishes a computational-to-experimental pipeline for discovering macrocyclic peptides that disrupt challenging protein-protein interactions and provides a foundation for developing next-generation SLIT2/ROBO1 modulators for cancer and neuroimmune disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/684696v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@e011f7org.highwire.dtl.DTLVardef@bb8e5aorg.highwire.dtl.DTLVardef@17ec46corg.highwire.dtl.DTLVardef@1919666_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

Structural and Biophysical Basis for PFAS Binding by Human Sterol Carrier Protein-2

Per- and polyfluoroalkyl substances (PFAS) are harmful environmental contaminants that bioaccumulate in human tissues and are linked to adverse health outcomes. While PFAS are known to bind to a variety lipid binding proteins (LBPs), such as human serum albumin and fatty acid-binding proteins (FABPs), the broader molecular basis for their biological distribution and additional target proteins in humans remains unanswered. Motivated by its known promiscuity towards a range of hydrophobic ligands, we investigated the interaction between human sterol carrier protein 2 (SCP2) and various PFAS. SCP2 is a structurally distinct LBP with no previously reported affinity for PFAS. Using a combination of screening, fluorescence displacement assays, protein structure prediction of PFAS-SCP2 complexes, ITC, and NMR experiments, we demonstrate for the first time that SCP2 is a PFAS-binding protein. Our findings provide insight into the residues participating in these interactions and provide evidence for an additional LBP that may facilitate PFAS distribution and persistence in the human body.

biophysics↗

Biophysical Design Space for Cellular Self-assembly and Dynamics

In natural biological systems, cells organize into tissues through interactions of several processes, including cellular signaling, collective migration, contractile activity of cytoskeletal elements and interactions with their surroundings. In recent decades, advancements in microscopy, genetic engineering, biochemistry, and computational modeling have enabled a more quantitative understanding of these processes. In this article, we present an integrated computational framework that couples various physical mechanisms such as cell-cell adhesion, strength and persistence of cellular motility, and the background stiffness. We use this framework to study how they collectively interact to determine the self-organization of the cells from a pseudo-random structure as well as the migration behavior of the cells. Notably, our simulations predict that motility has a two-way effect on cellular self-assembly: it promotes aggregation at moderate levels but disrupts clusters when excessively strong, yielding an optimal motility for formation of multicellular clusters. On the other hand, adhesion shows a two-stage effect: At lower value it self-assembles the structure, at higher value it compacts it. Furthermore, we experimentally demonstrate the motility-assisted self-aggregation of cells using cancer cells in a granular mechanical milieu. Finally we show that cell-cell adhesion and background medium tune the strength and persistence of cellular migration. Altogether, this work presents a computational framework that allows us to design phase behavior of collective of cells tuning their interaction, motility, and the background mechanics.

biophysics↗

A biophysical model of molecular clusters: interplay of multivalency, membrane localization, steric hindrance, molecular flexibility and intracellular crowding

Dynamic molecular clusters are assembled through weak multivalent interactions and are platforms for cellular functions, especially receptor-mediated signaling. Clustering is also a prerequisite for liquid-liquid phase separation. But it is not well understood how molecular structure and cellular organization control clustering. Using coarse-grain kinetic Langevin dynamics, we performed computational experiments on a prototypical ternary system modeled after membrane-bound nephrin, the adaptor Nck1 and the actin nucleation promoting factor NWASP. Steady state cluster size distributions favored stoichiometries that optimized binding (stoichiometry matching), but still were quite broad. At high concentrations, the system can be driven beyond the saturation boundary such that cluster size is limited only by the number of available molecules. This behavior would be predictive of phase separation. Domains close to binding sites sterically inhibited clustering much less than terminal domains because the latter effectively restrict access to the cluster interior. Increased flexibility of interacting molecules diminished clustering by shielding binding sites within compact conformations. Membrane association of nephrin increased the cluster size distribution in a density-dependent manner. These properties provide insights into how molecular ensembles function to localize and amplify cell signaling.

biophysics↗

Biophysical Studies of Some Bee Products as Radioprotectors

The study had been planned to evaluate some antioxidant ingredients in honey and propolis. Also, a study on ionizing gamma irradiated rats was done to assess these antioxidants as radioprotectors. Bioactive ingredients, such as phenols, flavonoids and trace elements, were explored using FTIR, UV-Vis and AAS spectroscopic techniques. Animals were exposed to fractionated gamma radiation doses. Honey, propolis and their combination were administrated before and during the irradiation period. Serum levels of total protein, albumin and uric acid were estimated. Also, the osmotic fragility of Red Blood Corpuscles (RBCs) membranes and a microscopic examination of blood films were investigated. The analysis demonstrated that the level of phenolic, flavonoid and trace elements are higher in propolis than honey. The levels of total protein and albumin decreased post irradiation while the level of uric acid increased. Likewise, osmotic fragility of RBCs membranes was increased with a sticking forming RBCs aggregation. It had been found that administration of the natural antioxidants induced amelioration in most of the studied parameters. It can be concluded that natural antioxidants produced a modulation against oxidative stress induced by ionizing radiation.\n\nSummary StatementAssessment of some antioxidant ingredients in honey and propolis. Also, a study on ionizing gamma irradiated rats was done to assess these antioxidants as radioprotectors.

biophysics↗

First-in-Class Small Molecule ROBO2 Binders Identified through Integrated Virtual Screening and Biophysical Validation

Roundabout homolog 2 (ROBO2) is a transmembrane receptor implicated in glioblastoma progression through its interaction with Slit2-mediated signaling pathways. Dysregulated Slit2-ROBO2 signaling enhances tumor cell migration, invasion, and tissue infiltration, while elevated ROBO2 levels contribute to an immunosuppressive tumor microenvironment supporting GBM aggressiveness and highlighting ROBO2 as a therapeutic target. Despite its therapeutic relevance, no ROBO2-targeted small molecules have been reported. To address this gap, we performed a structure-based virtual screening campaign targeting ROBO2, followed by experimental validation with Dianthus TRIC platform and microscale thermophoresis (MST). Fifteen compounds were screened for ROBO2 binding, from which four candidates exhibited robust TRIC signals. Subsequent affinity measurements revealed that two small molecules, Z1334432986 and Z1692774161, bind ROBO2 in a reproducible concentration-dependent manner, with dissociation constants (Kd) of 40.8{+/-}4.8 M and 25.8{+/-}16.95 M, respectively. Molecular docking with validated hits revealed a shared ROBO2 binding pocket defined by conserved anchor residues (ASN354, SER366, ASP385) and accommodation of distinct ligand conformations within the ROBO2 binding pocket. This work establishes a screening pipeline for identifying ROBO2-targeted small molecules and lays the foundation for developing therapeutics aimed at disrupting Slit-ROBO2 signaling in GBM. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/697640v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@2fa804org.highwire.dtl.DTLVardef@bfa3d5org.highwire.dtl.DTLVardef@19d1522org.highwire.dtl.DTLVardef@1d6774a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

Selective Stabilization of HRAS2 i-Motif DNA by TMPyP4: A Multimodal Biophysical and Thermodynamic Investigation

I-motif (iM) DNA structures, formed by cytosine-rich sequences, are increasingly acknowledged for their involvement in gene regulation, maintenance of genomic stability, and their emerging potential as therapeutic targets, particularly in cancer. Despite their biological relevance, the discovery of selective small-molecule probes that can specifically recognize and interact with iM DNA remains an ongoing challenge. In this study, we have used TMPyP4 and screened for its ability to bind various iM DNA constructs, including HRAS1, HRAS2, VEGF, CMYC, CKIT and H-Telo. Structure-activity relationship analyses revealed that specific substitution patterns conferred selectivity towards HRAS2 iM target. Comprehensive spectroscopic investigations, including UV-Vis absorption, steady-state and time-resolved fluorescence, and fluorescence anisotropy, uncovered key photophysical signatures of binding, including significant hypochromic and bathochromic shifts, enhanced fluorescence emission, and prolonged fluorescence lifetimes. Circular dichroism (CD),thermal denaturation (UV-melting) and thermodynamic investigations confirmed that TMPyP4 effectively stabilized the HRAS2 iM structures without disrupting their native topologies. Meanwhile, FT-IR spectroscopy revealed local structural rearrangements upon TMPyP4 binding, offering further evidence of molecular interaction. Collectively, these findings provide valuable insights into the molecular recognition of iM DNA by TMPyP4 and highlight its promise as both selective HRAS2 iM-binding agent and responsive fluorescent probe. This work lays a strong foundation for the development of novel tools for studying iM structures in biological systems and for designing future therapeutics targeting iM DNA in cancer and related diseases.

biophysics↗

Orthosteric and allosteric effects of anti-CRISPR II-C1 inhibition on GeoCas9 from integrated structural biophysics

Anti-CRISPRs (Acrs) are small protein inhibitors of CRISPR-Cas effectors that originate from the translated genetic material of bacteriophage. Harnessing the natural ability of Acrs to bind and disrupt CRISPR-Cas editing can provide enhanced spatiotemporal control of gene editing. Recent studies have revealed diverse structures and functions of Acrs, however, atomistic studies of the specific molecular mechanisms behind Acr inhibition are lacking. Here, we reveal how structure, function, and dynamics govern AcrIIC1 inhibition of Cas9 from G. stearothermophilus (GeoCas9) via its HNH nuclease domain. An X-ray crystal structure of the GeoHNH-AcrIIC1complex reveals a conserved binding interface at the catalytic site and disruption of crucial electrostatic contacts known to modulate the thermostability of GeoCas9. AcrIIC1 binding also rewires the intrinsic dynamics of the GeoHNH domain, stimulates millisecond motions that are absent from the unliganded nuclease, and attenuates the guide RNA affinity of GeoCas9. Subsequent AcrIIC1 mutations in residues at its crystallographic binding interface uncouple Acr binding from inhibition, providing new insight into mechanism by which AcrIIC1 acts on GeoCas9.

biophysics↗

Biophysical properties of material-colonizing fungal biofilms: multiscale structural and mechanical characterization

Black extremotolerant fungi form persistent biofilms on a wide range of natural and engineered substrates. Able to weather minerals, affect stone monument surfaces, and colonize solar panels, they demonstrate a strong capacity to interact with and modify material surfaces, even under most extreme conditions. In this study, we establish a methodological workflow for the structural and mechanical characterization of melanized biofilms formed by the black fungus Knufia petricola. This species represents a broader group of resilient surface colonizers and provides a model for in-depth investigation. When grown on solid agar/air interface, this species predominantly forms a compact biofilm, composed of spherical cells, while retaining the capacity for filamentous growth, providing a suitable framework to explore morphology-dependent biomechanical responses. The proposed toolbox combines complementary analytical techniques spanning multiple spatial scales, including shear-rheology to quantify bulk viscoelastic behavior, micro-indentation to resolve local stiffness of the biofilm surface, micro-computed tomography for non-destructive three-dimensional visualization of biofilm architecture, and cryogenic preparation methods and electron microscopy for high-resolution ultrastructural analysis. As a case study, we applied this workflow to compare biofilms grown on two nitrogen sources (NO3- vs. NH4+). Our results reveal that the nitrogen source plays a key role in biofilm morphology across multiple hierarchical levels - ranging from cell division patterns and distribution of extracellular polymeric substances (EPS) to overall mechanical properties, where NO3- leads to budding-dominated growth and increased stiffness, whereas NH4+ promotes meristematic growth and softer biofilms. The successful transfer and integration of methods originally developed for bacterial biofilm research highlights the feasibility of quantitative mechanical analyses in fungal systems. This multiscale toolbox provides a foundation for advancing the mechanistic understanding of fungal biofilms and biofilm-material interactions, with implications for geomicrobiology, material biodeterioration, and the design of bio-inspired functional materials. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/720134v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@7fd08borg.highwire.dtl.DTLVardef@15476c2org.highwire.dtl.DTLVardef@40be50org.highwire.dtl.DTLVardef@8e9410_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗