International Journal of Biological Macromolecules

Kyasanur Forest Disease Virus (KFDV) NS5 methyltransferase (MTase) protein is the essential enzyme that is involved in the cap methylation of viral RNA, viral replication, and immune evasion, and therefore it is an important protein of interest for antiviral research and drug design. In the present work, we successfully resolved the three-dimensional crystal structures of KFDV NS5 MTase co-crystallised with SAH and GTP at resolutions of 2.2 [A] and 2.6 [A], respectively. In previous studies, HC (Herbacetin) and CAPE (Caffeic acid phenethyl ester) have shown inhibitory activity against SAM-dependent viral MTase. To evaluate the inhibitory potential of HC and CAPE against KFDV NS5 MTase, we have performed isothermal titration calorimetry (ITC) and tryptophan fluorescence spectroscopy (TFS) to validate protein interaction with target compounds. MTase inhibition assay was performed using capillary electrophoresis (CE) assays. Additionally, fluorescence polarisation (FP) confirmed RNA binding inhibition by CAPE and HC. Together, these experiments suggest that HC and CAPE are promising inhibitors against KFDV NS5 MTase and could potentially act as lead compounds to design broad-spectrum anti-Orthoflavivirus drugs.

Amyloid fibrils are highly ordered protein aggregates characterized by a conserved cross-{beta}-sheet architecture despite originating from structurally diverse precursor proteins. Growing evidence suggests that interactions between different amyloidogenic proteins can modulate aggregation pathways through heterologous cross-seeding; however, the influence of seed polymorphism on the structure and biological properties of cross-seeded fibrils remains poorly understood. Here, we investigated the cross-seeding of native human insulin by two structurally distinct polymorphs of hen egg-white lysozyme (HEWL): flexible fibrils (FFs) and rigid fibrils (RFs). Native insulin remained stable under physiological conditions and underwent spontaneous fibrillation only under acidic conditions. In contrast, both HEWL polymorphs efficiently induced insulin aggregation at physiological pH, bypassing the nucleation barrier. Thioflavin T fluorescence, circular dichroism spectroscopy, and transmission electron microscopy revealed that lysozyme FFs templated the formation of insulin flexible fibrils (IFFs), whereas lysozyme RFs produced insulin rigid fibrils (IRFs), demonstrating that the structural characteristics of the parental HEWL polymorphs were propagated during heterologous cross-seeding. The toxicity of the resulting insulin fibrils was evaluated in SH-SY5Y neuronal cells and CCF-STTG1 astrocytes. IFFs exhibited minimal cytotoxicity and only subtle morphological alterations, whereas IRFs caused modest reductions in cell viability accompanied by more pronounced cellular damage. These findings demonstrate that the structural polymorphism of HEWL fibrils governs both the architecture and biological activity of cross-seeded insulin fibrils, highlighting amyloid polymorphism as an important determinant of heterologous amyloid propagation and a potential design principle for engineering functional amyloid-based biomaterials and protein delivery platforms.

Antifreeze proteins (AFPs), lower the freezing point via thermal hysteresis activity and/or ice recrystallization inhibition, playing a crucial role in protecting organisms from freezing damage under sub-zero milieu. This property endows them with promising applications in biomedicine and agriculture, ranging from tissue-organ cryopreservation to the development of frost-resistant crops. However, the lack of comprehensive resources dedicated for AFPs hinders further progress in elucidating their functional mechanisms and advancing their applications. Here, we report AFP-R, an online resource comprising AFP-DB and AFP-Predictor. AFP-DB is a comprehensive database with manually curated proteins bearing experimentally validated antifreeze activity derived from published literature, whereas AFP-Predictor is a sequence-based machine-learning model to identify AFPs. AFP-DB stores diverse AFP-related information, including sequences, structures, post-translational modifications, taxonomy and annotations of antifreeze-activity experimental assays. It now holds 186 entries, 607 sub-entries, and 1444 experimental records. AFP-Predictor, an AFP-identification algorithm built on protein language model ESM2 (Evolutionary Scale Modeling2), is trained on data in AFP-DB and outperforms several existing models. This work offers a valuable resource for systematically dissecting the mechanisms underlying AFP antifreeze activity and will facilitate their broader applications.

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), a key regulator of mitophagy, has been linked to the pathogenesis of Parkinson's disease (PD). PINK1 recruits Parkin, an E3 ubiquitin ligase, triggering mitophagy in response to mitochondrial damage. During mitophagy, the quantity, stability, and activity of PINK1 must be strictly regulated; however, the mechanisms governing these parameters under cellular stress are still unclear. Herein, we determined the structural basis for PINK1 maturation mediated by heat shock protein 90/cell division cycle 37/FK506-binding protein 51 (HSP90/CDC37/FKBP51) chaperone complex. We identified PINK1-associated proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and determined the structures of the complexes using Cryo-Electron Microscopy (Cryo-EM). Results showed that FKBP51 potentially interacts with a conserved leucine-proline-phenylalanine (LPF) motif on the activation loop of PINK1 and negatively regulates PINK1 functions in mitophagy. A PINK1 mutation located at the FKBP51 recognition site is linked to mitophagy deficiency, which can be partially rescued by specific inhibition of FKBP51. These findings reveal a general mechanism for PINK1 recognition by the HSP90/CDC37/FKBP51 chaperone complex and suggest a potential approach for upregulating PINK1 activity, which is impaired in PD.

Nisin is a promising antimicrobial peptide widely used in food preservation due to its efficacy against Gram-positive spoilage and pathogenic bacteria. Although Nisin is increasingly applied in the food sector, the biopeptide suffers from instability within food matrixes and can rapidly lose its antimicrobial potential following interaction with food biomolecules. Thus, it is necessary to investigate approaches that can be employed to extend the stability and activity of Nisin. Hence, the aim of this study was to develop and characterise a chitosan-alginate polyelectrolyte microencapsulation system capable of enhancing Nisin stability while retaining antimicrobial activity. The microencapsulation of Nisin was achieved by pre-gelation of alginate using calcium chloride and subsequent direct electrostatic interaction between cationic Nisin and chitosan with pre-gelled anionic alginate at pH 5.0. Following microcapsule formation, physicochemical and structural characterisation was performed using Zeta potential determination and measurement of the polydispersity index (PDI) via dynamic light scattering. SEM micrographs were used to confirm morphology, while Fourier-transform infrared (FTIR) spectroscopy and high-performance liquid chromatography (HPLC) were utilised to assess chemical integrity and functional group preservation of encapsulated Nisin. Following this, stable microcapsules with diameters ranging from 150-200 nm and smooth surface morphology were obtained. Microcapsule formation was strongly influenced by formulation parameters, particularly pH, calcium ion concentration, and chitosan content, with deviations from optimal acidic conditions (< pH 5.0) resulting in aggregation, increased polydispersity, and reduced encapsulation efficiency. The microcapsules were monodispersed (PDI {approx} 0.30) and electrostatically stable, exhibiting a Zeta potential of approximately +36 mV. These microcapsules remained stable over a prolonged storage period of 21 days under refrigerated conditions while retaining antimicrobial activity against Bacillus cereus. Encapsulation efficiency reached approximately 65%, confirming effective retention of Nisin within the polymer matrix. Overall, the findings demonstrate that chitosan-alginate ionic gelation is a non-denaturing and effective encapsulation strategy for extending the functional stability of Nisin. These microcapsules show strong potential as natural antimicrobial delivery systems for food and beverage applications, particularly in acidic food matrices, with implications for improved food safety and shelf-life extension.

The High Mobility Group Box 1 (HMGB1) protein performs multiple essential functions in the body, ranging from DNA regulation to the activation and mediation of immune responses. However, HMGB1 has been also implicated in several pathological conditions, such as rheumatoid arthritis, sepsis, autoimmune diseases, tumors, and Alzheimer's disease. As a result, HMGB1 is of increasing interest as a therapeutic target. Binding to heparin has been reported to inhibit HMGB1's pathological activity during sepsis in clinical settings. In this work, we compare the interactions of HMGB1 with heparin and its' synthetic analog linear polyglycerol sulfate (lPGS) from the viewpoint of stability and changes to association behavior. This analysis focuses on thermal stability, secondary-structure changes, and particle-size evolution using nano-differential scanning fluorimetry (nanoDSF), circular dichroism spectroscopy (CD), and dynamic light scattering (DLS).

Mucopolysaccharidosis IIIC (Sanfilippo syndrome type C) is a rare lysosomal storage disorder caused by loss-of-function mutations in HGSNAT, which encodes an enzyme involved in heparan sulfate (HS) degradation, leading to impaired HS catabolism, lysosomal accumulation, and progressive neurodegeneration. Because enzyme replacement therapies have limited penetration across the blood-brain barrier, substrate-reduction therapy represents an alternative therapeutic strategy. Here, N-deacetylase/N-sulfotransferase 1 (NDST1), a key enzyme responsible for HS biosynthesis, was investigated as a potential substrate-reduction target. A structure-based computational pipeline was used to identify and evaluate inhibitors targeting the NDST1 sulfotransferase domain. Approximately 4.1 million drug-like compounds and FDA-approved drugs were screened by molecular docking, followed by pharmacokinetic filtering, molecular dynamics simulations, and MM/PBSA binding free energy calculations. In parallel, peptide binders targeting the same site were generated using diffusion-based protein design and evaluated using molecular dynamics and MM/GBSA analysis. Four chemically distinct small-molecule scaffolds and three peptide candidates were identified as stable binders to the NDST1 active site. The lead small-molecule candidate exhibited a predicted binding free energy of -13.36 {+/-} 5.87 kcal mol-1. These provide a focused set of candidates for further investigation and support the feasibility of targeting NDST1 as a substrate-reduction strategy for MPS IIIC.

Cyclic oligosaccharides possess industrial advantages, including molecular encapsulation capability and high physicochemical stability, owing to the absence of a reducing end. Recently, a novel cyclic tetrasaccharide, cycloisomaltotetraose (CI4), consisting of four -1,6-linked glucose units, and the enzymes responsible for its synthesis, cycloisomaltotetraose glucanotransferases (CI4Tases), were discovered. Unlike known cycloisomaltooligosaccharide glucanotransferases (CITases) that yield a wide distribution of cyclic products with a degree of polymerization (DP) of 7 or higher, CI4Tases strictly produce CI4. To elucidate the molecular mechanism underlying this strict DP4 specificity, we determined the crystal structures of CI4Tase from Agreia sp. D1110, in its ligand-free form, as well as in complex with the linear hydrolysis product isomaltotetraose (IG4) and with CI4. Structural comparisons revealed that a loop (M247 to R251) blocks the region corresponding to the -5 subsite of typical CITases, narrowing the substrate-binding pocket. This "molecular ruler" mechanism ensures that only a glycan chain of exactly four glucose units is accommodated for cyclization. Among mutants of the residue positioned at the center of bound CI4, the formation of by-products other than CI4 was significantly suppressed in F245L, F245A, and F245W. While the cyclization activity of all F245 mutants decreased, the CI4 hydrolysis activity of these three mutants was also significantly reduced, resulting in an increased specificity for cyclic sugar production. These findings elucidate the strict size-control mechanism of CI4Tase and provide a structural foundation for engineering cycloisomaltooligosaccharide-producing enzymes with optimized transglycosylation efficiency and specificity for industrial applications.

Plant-derived small molecules possess highly diverse physicochemical properties, and the computational design of their protein recognition elements depends not only on the global structural quality of candidate backbones, but also on whether the local binding pocket, ligand-contact pattern, and predefined recognition conformation can be consistently retained after sequence design and structural back-prediction. To explore pocket-design strategies for different types of natural-product small molecules, this study selected capsaicin, (4R)-limonene, and quercetin as model ligands, representing a flexible amphipathic molecule, a compact hydrophobic monoterpene, and a rigid polyphenolic flavonoid scaffold, respectively, and covering the dimensions of pungent sensory flavor, volatile aroma, and flavonoid functional constituents. A ligand- physicochemical-property-guided computational design and multi-stage prioritization framework was established for candidate protein binders. The results showed that candidates with favorable initial global structural scores did not necessarily form reasonable local small-molecule binding pockets, indicating that evaluation of the local ligand environment is essential for candidate prioritization. After screening, 31 partial- pocket candidate backbones for capsaicin, 75 buried hydrophobic-pocket candidate backbones for (4R)-limonene, and 56 pocket-qualified candidate backbones for quercetin were obtained. Further sequence design and structural back-prediction analyses indicated that a subset of candidates could maintain the original pocket geometry and major ligand-contact patterns after sequence realization. Overall, these results suggest that the physicochemical properties of different plant-derived small molecules substantially influence the efficiency of de novo protein pocket formation, with compact hydrophobic ligands being more compatible with buried hydrophobic- pocket strategies, whereas flexible or multipolar ligands require a more refined balance between hydrophobic burial and polar exposure. This study provides a pre- experimental computational prioritization framework for natural-product small- molecule-recognizing proteins and offers candidate resources for subsequent protein expression, in vitro binding validation, active-constituent enrichment, and development of small-molecule biorecognition tools. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/743643v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@8fe6c2org.highwire.dtl.DTLVardef@176cef2org.highwire.dtl.DTLVardef@10c8201org.highwire.dtl.DTLVardef@2b28cf_HPS_FORMAT_FIGEXP M_FIG C_FIG

A protein whose removal or deletion causes significant disruption or collapse of a protein-protein interaction (PPI) network is referred to as a vulnerable protein. Such proteins may serve as valuable therapeutic or diagnostic targets in disease-associated networks. In this study, two PPI networks were constructed, one for HPV-positive and the other for HPV-negative head and neck squamous cell carcinoma (HNSCC), and the vulnerable proteins of these networks were identified by the node deletion approach. After analyzing the networks, 27 unique vulnerable proteins in HPV-positive and 72 unique vulnerable proteins in HPV-negative HNSCC were identified. Among them, one HPV-positive and seven HPV-negative HNSCC vulnerable proteins were further chosen by integrating multi-omics data. To exploit the vulnerabilities of these proteins, candidate synthetic lethal (SL) partners were predicted whose inhibition may selectively impair tumor survival. Subsequently, drug-gene interaction analysis was performed to identify inhibitors targeting the SL partners of these vulnerable proteins. Notably, in HPV-positive HNSCC, TOP2A, CHEK1, and CHEK2 genes were identified as SL partners of TTN, and their inhibitors were already clinically approved. While in HPV-negative HNSCC, ADA and MMP19 were identified as an SL partner of LMO7; TMEM45B, CDH3, and ELF3 genes were identified as an SL partner of CGN; and ZNF433 was identified as an SL partner of FLNC. However, MMP19, ZNF433, and TMEM45B inhibitors were not reported. Thus, these vulnerable proteins, including their SL partners, provide novel avenues to explore and develop more efficient and precise therapeutic and diagnostic strategies.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by progressive motor neuron degeneration. Mutations in the SOD1 gene represent the second most common genetic cause of ALS (ALS), and distinct SOD1 missense variants present with markedly different clinical profiles. A4V leads to an aggressive form of the disease (median survival [~]1y), H46R confers a mild, slowly progressive course and I113T exhibits an intermediate phenotype. The molecular basis by which these mutations produce divergent clinical outcomes remains poorly understood. We performed extensive classical molecular dynamics simulations of wild-type SOD1 and the three ALS-associated variants in the apo monomeric state to attempt to investigate the mechanisms behind such phenotypic differences. Structural stability, global compactness, and conformational flexibility, as well as analysis of collective motions between residues and estimation of free energy, were assessed. The H46R, A4V, and I113T variants exhibited distinct dynamic behaviours, highlighting differences in structural stability, local flexibility, and intramolecular interactions. These findings suggest that specific structural regions may contribute differently to protein dysfunction and could represent key elements for understanding the relationship between molecular dynamic properties and the differing clinical severity associated with these variants. Most strikingly, H46R exhibited exceptional structural stability across every analytical level, the lowest global deviation, most attenuated local flexibility, strongest internal dynamic coordination, and the deepest, most confined free energy basins of any system examined. This convergent multi-layered evidence of structural restraint provides a compelling mechanistic basis for the mild and slowly progressive clinical course of H46R ALS, suggesting that enhanced conformational rigidity, rather than bulk destabilisation, is the defining biophysical feature of this variant, and that its pathogenic mechanism operates through a route fundamentally decoupled from the aggregation-driven toxicity that characterises the more aggressive SOD1-ALS mutations.

Kinema is a traditional fermented soybean food indigenous to the eastern Himalayan regions of Nepal and India. The fermentation process is primarily mediated by the bacterium Bacillus subtilis, which produces several bioactive compounds and enzymes with potential therapeutic applications. Considering the growing burden of cardiovascular diseases and the need for effective fibrinolytic agents for thrombolytic therapy, this study aimed to extract, partially purify, and evaluate the thrombolytic potential of kinemakinase derived from kinema prepared from white soybeans. Partial purification of the enzyme was achieved using ammonium sulfate precipitation. Thrombolytic activity was assessed in vitro using human blood clots, where three enzyme dilutions demonstrated clot lysis ranging from 66% to 68%, indicating considerable fibrinolytic potential. In silico analyses were also performed to investigate the structural and functional characteristics of the enzyme. The tertiary structure obtained from UniProt was modeled using the Robetta server and refined with GalaxyRefine. Docking with fibrin using ClusPro 2.0 and molecular dynamics simulations using iMODS confirmed favorable interaction and structural stability, while disulfide engineering enhanced protein stability. The findings suggest that kinema-derived kinemakinase may serve as a promising alternative thrombolytic agent, warranting further biochemical characterization and dosage optimization.

Protein engineering and precision fermentation provide an opportunity to increase the value of food proteins by improving their solubility, stability, functionality, or nutritional composition. Here, we use {beta}-lactoglobulin ({beta}LG) as a model protein to investigate how state-of-the-art computational protein design approaches affect these properties. First, the deep learning-based design tool ProteinMPNN was used to alter up to 20% of {beta}LG residues for increased stability. Second, the physics-based modeling platform PyRosetta was used to find positions in {beta}LG accommodating increased branched-chain amino acid (BCAA) content and up to 10 residues were simultaneously exchanged. Experimental characterisation of ProteinMPNN and stabilised BCAA-enriched variants showed similar secondary structure and oligomeric state as native {beta}LG. ProteinMPNN variants gave increased titers and increased thermal stability up to 15 {degrees}C, and this correlated with changes in the rate of surface pressure in droplet tensiometry. Stabilized BCAA-enriched mutants had altered acid solubility. Correlations between computationally derived biophysical metrics and experimental properties are presented and suggest some predictive power for surface hydrophobicity on protein yield.

The binding affinities of a chimeric analog of a riboflavin derivative linked to biotin, (6- (7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexyl 5-((3aS,4S,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), referred to as C6-Rf-biotin-tag, to the riboflavin binding retain or streptavidin are in the M range, 1.29 {+/-} 0.277 and 3.00 {+/-} 0.459, respectively. These values suggest that C6-Rf-biotin-tag has potential applications in diagnostic assay and labelling target flavin binding proteins. The C6-Rf-biotin-tag which was characterized with respect to physical and biochemical properties retains UV/Vis spectroscopic and fluorescence behavior similar to riboflavin.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated nuclease gene (Cas), originating from the bacteria acquired immune system, have revolutionized gene editing technology. In this regard, type II (Cas9) been extensively studied and widely applied CRISPR system so far. The mechanism for precise manipulation of genomic sequences is guided by small RNA called CRISPR RNA (crRNA). In this study we devised and optimized CRISPR-Cas9 screening system based on Cas9 gene detection, targeting a conserved part of recognition domain (REC) consisting of arginine rich bridge helix (BH). We used hemi-nested PCR approach for screening sensitivity and reproducibility. The recombinant E. coli DH5 alpha containing the pRGEB32 vector (DH5 alpha/pRGEB32) with the Cas9 gene was used for system optimization. Subsequently, the screening system was applied and validated on different environmental bacterial strains including Alcaligenes faecalis and Pseudomonas stutzeri, isolated from sewerage samples. The optimized hemi-nested PCR resulted in amplification of targeted region in environmental bacterial strains and results were reproduced successfully. Furthermore, nucleotides and amino acid sequence, motif and domain analysis of PCR products, confirmed the targeted Cas9 REC-BH domain. Presently, no rapid and cost effective CRISPR-Cas screening system is available except expensive whole genome sequencing approach. Our investigation aimed to device rapid and cost effective screening system for identification of new variants of Cas9 proteins in environmental bacterial species. In this context, the developed Cas9 gene-based CRISPR-Cas screening system (C9CSS) may be a potential rapid screening tool to identify new Cas9 orthologs in different bacterial genomes with improved functions.

Per-and polyfluoroalkyl substances (PFAS) constitute a diverse class of persistent synthetic chemicals utilized across industrial, medical, and consumer sectors that are pervasive global pollutants. Exposure to PFAS is linked to adverse impacts on both innate and adaptive immune systems. Human lactoferrin (hLF) is a key antimicrobial component of the developing innate immune system present in colostrum and breast milk. We hypothesized that hLF is a potential PFAS binding protein related to PFAS immunotoxicity. The results of thermal stability experiments indicated that all 11 tested PFAS bind and destabilize the structure of hLF. Notably PFBA, PFOS, HFPO-DA, and 6:2 FTSA decreased apo-hLF melting temperatures from 64oC to [&le;] 37oC, suggesting that PFAS exposures destabilize the native hLF protein under physiological conditions. Relative binding affinities (Kd) ranged from 0.2-11 mM across tested PFAS. Molecular docking was used to confirm experimental binding affinities and identify molecular interactions involved with PFAS binding. Calculated Gibbs Free Energies of binding ranged from -4.4 to -8.8 kcal/mol. Together, these results demonstrate that PFAS bind hLF at affinities comparable to human serum albumin and other PFAS binding proteins, and that some PFAS can destabilize hLF protein structure at physiologically relevant temperatures and conditions.

We present the solution structures of HIV-1 proteins NC(p7)1-55 corresponding to the full-length NC(p7) and mature p6. The studies were carried in water and, to mimic the membrane, in micellar DPC (Dodecylphosphocholine) conditions. Our results unravel for the first time the structure adopted by the N-terminal amino acids of the free NC(p7)1-55, with the formation of a small helix spanning residues F6 to R10. Our NMR and Fluorescence Anisotropy data disclose an interaction between NC(p7)1-55 and p6 both in water and DPC, with respective Kd of 2.5mM and 370 mM at 23{degrees}C. The interaction is thus strengthened in lipidic conditions. Protein p6 stabilizes the N-terminus of NC(p7)1-55 while increasing at the same time the dynamic of the first zinc finger. Although the entire p6 sequence is involved in the interaction, we show that its C-terminal region is particularly sensitive to the presence of NC(p7)1-55, with a propensity of forming a a helix ranging from amino acids S111 to F116. This study brings experimental evidence of a direct protein-protein interaction between p6 and the N-terminal region of NC(p7)1-55. We further show that such interaction is readily accommodated within the NC(p15) framework and hypothesize that it may facilitate the selective assembly of assembly of the viral genomic RNA (gRNA) in the cell.

Every known life form senses and reacts to mechanical forces. These mechanical stimuli can be converted into electrical signals by mechanically gated ion channels, a transduction cascade pivotal to numerous physiological functions including touch, hearing, mechanical pain, circulation, gastrointestinal function, and mechanical loading in various tissues. Despite continuous efforts, numerous mechanically gated ion channels with the mechanotransduction process underlying these physiological functions remain unidentified. Here, we focused on the transmembrane channel-like (TMC) protein family expressed in the cultured cells to identify those with potential mechanosensitive activity. Remarkably, in contrast to human TMC1/2 (HsTMC1/2), human TMC3-8 (HsTMC3-8) proteins are localized to the plasma membrane when heterologously expressed in the cultured cells. Further experiments revealed that mechanical poking stimuli can effectively activate HsTMC3-8. In addition, HsTMC3-8 induced stretch-activated currents and elicited well-resolved single-channel activities in response to negative pressure stimulation. The mutants near the putative pore region altered reversal potentials (Erev) of HsTMC3-8, suggesting that TMC3-8 are likely pore-forming subunits of ion channels. In summary, we proposed that TMC proteins are the largest mammalian mechanically gated ion channel family.

Hydrophobins are biosurfactant proteins that coat the cell surfaces of filamentous fungi. On the conidial surface, hydrophobins self-assemble into rodlets, forming a dense hydrophobic film that promotes air-dispersibility. Although rodlet formation is closely associated with the physiology of filamentous fungi, its underlying molecular mechanisms remain largely unknown. Previously, we revealed that RolA, a hydrophobin derived from Aspergillus oryzae, forms rodlets at the air-water interface. In this study, we focused on the flexible N-terminal region of RolA, which lacks a well-defined tertiary structure, and hypothesized that this intrinsically disordered region regulates rodlet formation. To investigate its role, we used RolA mutants with reduced charges in the N-terminal region and analyzed the rodlet formation process on the surface of a water-in-air sessile droplet using atomic force microscopy. In addition, we quantitatively characterized rodlet formation at the air-water interface by applying a kinetic perspective to the interfacial tension change profiles obtained from dynamic surface tension measurements. The results suggested that RolA first forms a monolayer at the air-water interface, then rodlet formation proceeds through the continuous supply of free RolA monomers from the bulk phase to the interfacial RolA film. Our molecular dynamics simulations of RolA at the interface supported a model in which RolA molecules within the interfacial film interact with free monomers in the bulk phase through their N-terminal regions. These results reveal a previously unidentified role of the N-terminal region in rodlet formation and provide a more comprehensive framework for understanding the molecular mechanism underlying RolA rodlet formation.

Circular single-stranded DNA (cssDNA) is a versatile biomolecule with applications spanning genome editing, DNA nanotechnology, synthetic biology, molecular diagnostics, and aptamer development. Compared with linear single-stranded DNA, cssDNA offers enhanced structural stability, resistance to in-cellulo degradation by exonucleases and enables the generation of long, sequence-defined DNA molecules that are difficult to obtain through conventional chemical synthesis methods. Despite its growing utility, widespread adoption of cssDNA has been limited by the lack of accessible, scalable, and cost-effective production methods, with many existing workflows relying on specialised reagents, extensive optimisation, or commercially synthesised DNA. Here, we present a streamlined, end-to-end protocol for the laboratory-scale production of high-purity cssDNA using an M13 phagemid-based system and standard molecular biology laboratory infrastructure. The workflow encompasses bacterial culture, phage amplification, nuclease treatment, phage precipitation, anion-exchange purification, and quality control, with practical optimisations to improve yield, reproducibility, and scalability. Using this approach, yields range from 120-195 {micro}g of purified cssDNA from 300 mL of culture supernatant. The protocol provides detailed guidance on critical process parameters, troubleshooting, and quality assessment, enabling reliable production of cssDNA suitable for a wide range of downstream molecular biology and genome engineering applications.