European Biophysics Journal

Protein oligomerization equilibria are central to many biological processes and are often highly sensitive to environmental conditions such as ionic strength, pH, and ligand binding. Quantitative characterization of these equilibria remains experimentally challenging because stable protein complexes frequently dissociate only at concentrations that are difficult to access with conventional biophysical methods. Fluorescence correlation spectroscopy (FCS) is uniquely suited to this problem, as it provides direct access to diffusion coefficients of fluorescently labeled proteins at nanomolar concentrations. However, the quantitative interpretation of FCS data from oligomeric systems requires a rigorous mathematical framework and careful experimental practice that have not previously been described in sufficient detail to guide implementation. Here, we provide a comprehensive description of the experimental workflow and analytical framework for determining dissociation equilibrium constants by FCS, covering instrument calibration, sample preparation, data quality control, after-pulse correction, and nonlinear least-squares fitting. We discuss common sources of error and provide practical guidance on critical experimental considerations including surface passivation, buffer preparation, equilibration time, and the role of labeling efficiency. Using the homotrimeric sliding clamp PCNA as a model system, we demonstrate the complete workflow under a range of KCl concentrations and show that moderate ionic strength stabilizes the PCNA trimer while very high salt partially destabilizes the complex. The approach is general and applicable to any reversible protein self-association reaction accessible by fluorescence detection at low protein concentrations.

Molecular dynamics simulations of nucleic acids are performed using a solvent-buffer distance of 10 [A] between the solute surface and the simulation box boundary. Although this cell size has been extensively explored in protein simulations, its implications for nucleic acid dynamics are not well understood. Nucleic acids are elongated, highly charged, and flexible structures with hydration and dynamical properties distinct from those of proteins and therefore, they may require different solvent-layer considerations in simulations. In this study, we investigated the effect of simulation cell size on nucleic acid dynamics by simulating a 30-base-pair double-helical nucleic acid structure and its two single-stranded forms using solvent-buffer distances of 3, 5, 10, 15, and 20 [A]. Smaller cells may impose restricted hydration, molecular crowding, and periodic image interactions. However, larger cells provide solvent space for conformational relaxation. A total of 45 s of molecular dynamics simulations were performed (3 structures x 5 cell sizes x 3 replicates x 1 s). Our results show that while the commonly used 10 [A] buffer may be sufficient to maintain the stability of the double-stranded nucleic acid, larger cells are required to capture the conformational dynamics of single-stranded structures. In both, increasing the cell size to 15 or 20 [A] enables broader conformational sampling. The first hydration shell exhibits reduced crowding in the 20 [A] cell, consistent with more relaxed conformations. At larger cell sizes, single-stranded nucleic acids adopt compact, self-associated conformations for stability. Together, this study presents physical insight into how simulation cell size and solvent environment influence nucleic acid dynamics.

Poly-L-Lysine (PLL) mediates the non-specific adhesion of cells and is commonly used in Atomic Force Microscopy (AFM) measurements, to ensure that cells remain attached to the substrate. However, it is acknowledged that adhesion affects the measured mechanical properties, in particular in the case Red Blood Cells (RBCs). This results in a wide range of Youngs modulus E reported in the literature. The present study aims at providing a systematic approach to the impact of non-specific adhesion on the rheology of RBCs. It provides a correlation between the topography profile of adherent RBCs and their rheology, from weak (cPLL = 10-3 mg/mL) to strong-adhesion (cPLL = 100 mg/mL) regimes. Using RICM and AFM, we find that there is a continuum of RBC shapes promoted by adhesion, from concave to dome-shaped, as predicted by the theory of vesicle adhesion. Their elastic properties discriminate them into two populations depending on adhesion strength, where stiffer RBCs (E {gtrsim} 100 Pa) correlate with dome-shaped cells. These findings are supported by rheology measurements of the dynamic complex shear modulus G*(f): while the storage modulus increases with cell-substrate adhesion, reflective of an increased membrane shear modulus, the loss modulus remains unchanged. Finally, further analysis inspired by membrane theory shows that different deformation modes may be triggered during indentation of either weakly or strongly adhering RBCs, illustrating the limits of the Hertz model.

The widespread use of synthetic food dyes, such as Acid Yellow 23 (AY 23), in the food, cosmetics, and pharmaceutical industries raises questions about their potential effects on biological systems and public health. The concentration-dependent interaction between AY 23 and bovine serum albumin (BSA), a crucial model protein for understanding pharmacokinetics and protein-ligand behaviour, was examined in this study. We demonstrate that, under physiological conditions, increasing dye concentrations from 50 M to 200 M results in notable conformational changes, increased surface hydrophobicity, and protein aggregation using a multimodal biophysical approach that includes fluorescence spectroscopy. Direct visualisation verified these structural changes and aggregate formation, whereas hemolytic assay confirmed the high hemolytic nature of AY 23-induced fibrils. Additionally, this study provides a mechanistic basis for the toxicological effects of AY 23, underscoring the implications of food dyes for public health.

Botanical dietary supplements (like wheat, barley, teff, oats, white lupin, pumpkin seed, and chickpeas) may contain trace amounts of toxicants in addition to important micronutrients. Developing and validating a reliable protocol for the simultaneous quantification of Cu, Fe, Zn, Mo, Se, Mn, Pb, Al, Ni, and Cr using a PerkinElmer (NexIONTM2000 model) quadrupole ICP MS (including a He collision and reaction cell when needed) with closed vessel microwave digestion using (HNO3 + H2O2) was the aim of this study.The method was subsequently utilized in a sample survey, and the outcomes were evaluated against WHO/JECFA standards. From five study regions, twenty-seven farm-collected botanical powder samples representing seven species were acquired. To create one composite per species, field subsamples were cleaned, air dried, ground, and blended (nine subsamples per botanical: three grabs from each of three farms). HNO3/H2O2 was used to digest aliquots (0.250-0.500gm) in closed microwave containers. Internal standards, multi-point external calibration, procedural blanks, verified reference materials, matrix spikes, and duplicates were all used in ICP MSs multi-element quantitation. Method LODs/LOQs, accuracy (CRM recoveries), and precision (RSD) were calculated.The technique produced low LODs that were suitable for dietary evaluation (typical LOD ranges: Cu, Fe, Zn, Mn, Ni, Cr (0.001-0.01) mg/kg; Mo, Se, Pb, Al (0.002-0.05) mg/kg. For the majority of analytes, within-run RSDs were less than 5%, while CRM recoveries ranged from 88.9 to 110%. The concentrations of essential elements varied greatly (average mg/kg: Fe (280.7{+/-}25.6); Zn (6.0{+/-}0.541); Cu (2.8{+/-}0.269); Mn (398.3{+/-}23.8); {micro}gm/kg: Se (0.061{+/-}0.006); Mo (1.0 {+/-}0.022). Although some composites approached or exceeded conservative intake thresholds for Pb and Al under high consumption scenarios, toxic elements were generally low (mean mg/kg: Pb (0.062{+/-}0.007); Al(185.2{+/-}18.5); Ni(1.6{+/-}0.163); Cr(1.8{+/-}0.171).For the simultaneous nutritional and contaminant profiling of supplements derived from cereals and those not, the validated ICP- MS workflow with microwave HNO3 and H2O2 digestion is suitable. Accurate labeling and consumer safety can be supported by routine screening and supply chain controls.

Small-angle X-ray or neutron scattering (SAXS/SANS) analysis of large unilamellar vesicles (LUVs) is often limited by high-dimensional bilayer models and the lack of dedicated, statistically rigorous workflows. Here, we introduce SAS_MoCa, an open-source Python package that integrates a compositional scattering density profile (SDP) description of lipid bilayers with a separated form factor (SFF) treatment of vesicle size and polydispersity, and couples these highly parameterized models to an adaptive thermodynamic simulated annealing algorithm formulated within a constrained Bayesian framework. SAS_MoCa enables users to incorporate quantitative prior information from, e.g., previous SAXS/SANS studies, dynamic light scattering, NMR, or molecular simulations, and returns full posterior parameter distributions, uncertainties (reported as medians and median absolute deviations) and correlations even from single SAXS curves. Validation on POPC, POPE and DMPC SAXS-only data demonstrates that the method yields reproducible structural parameters with uncertainties comparable to joint SAXS/contrast-variation SANS analyses. The modular architecture of SAS_MoCa facilitates extension to additional lipid systems and future joint SAXS/SANS or SANS-only applications.

Underwater adhesion research increasingly draws on bioinspired systems to uncover the molecular mechanisms that enable strong interfacial binding in aqueous environments. The biofilm adhesin Bap1 from Vibrio cholerae contains a short peptide motif, SYWFFGWHTK (CP), which exhibits exceptional adhesive performance, surpassing mussel foot protein mfp5 under comparable conditions. Despite its promise, the roles of ionic environments and aggregation behavior in governing CP adhesion remain unclear. In this study, we investigate how ion identity influences CP aggregation, film formation, and interfacial properties. Using dynamic light scattering, we identify the formation of micron-scale assemblies of aggregated molecular clusters (AAMCs), with size distributions modulated by salt type. Quartz crystal microbalance with dissipation and liquid atomic force microscopy reveal that CP film formation is both surface- and ion-dependent. On gold substrates, AAMCs preferentially adsorb and collapse into rigid, smooth nanofilms, consistent with hydrophobic-driven compaction. In contrast, silicate surfaces inhibit such collapse, yielding distinct morphologies and interfacial energetics. These findings demonstrate that surface chemistry and ionic conditions jointly regulate peptide aggregation and adhesion. This work provides mechanistic insight into hydrophobic-rich peptide systems and informs the rational design of next-generation wet adhesives, with broader implications for biomaterials and peptide-based formulations. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/733527v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1bd012aorg.highwire.dtl.DTLVardef@1977892org.highwire.dtl.DTLVardef@16cf79borg.highwire.dtl.DTLVardef@f405bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

For structural-biology and protein-production pipelines, the hardest part of a difficult protein is not the biology -- it is obtaining a well-behaved sample for functional studies. Programs routinely stall at construct design, expression, and purification: deciding where to truncate, which tags to use, how to express, and how to purify so the protein survives concentration and handling. These decisions are still made largely by literature precedent and experimental experience, and they require trial-and-error before arriving at a functional construct for hard targets. We present a prospective, single-pair wet-lab case study testing whether an integrated computational platform can improve these decisions. For human fibroblast growth factor 21 (FGF21) -- a clinically important and stability-challenged metabolic hormone -- we compared two expression constructs produced side by side under the same experimental workflow, using two different design strategies: one designed by a scientist from the literature (reproducing the published core-domain construct, PDB 6M6E), and one designed by the Orbion platform -- an AI, prediction-guided protein-design system (orbion.life) -- which additionally generated the expression and purification protocols (executed scientist-in-the-loop). The platforms construct used an unconventional, longer C-terminal boundary not found in public sequence databases. Since the two constructs differ in more than one feature, we treat them as workflow-level designs throughout. The scientist construct gave a higher initial yield ([~]2.4 xmore protein recovered at affinity capture). The platform-designed construct, however, showed a more favourable downstream developability profile: it concentrated higher (1.4 vs 0.7 mg/mL) while remaining more monodisperse by dynamic light scattering (DLS). The scientist construct, in contrast, aggregated on concentration, so its initial-yield advantage did not survive: in the final concentrated sample the Orbion construct provided the more usable material for downstream studies. Computed for the mammalian host used, the platform had prospectively scored its own design higher (composite 68.7 vs 59.0 for the scientist-designed construct), and its predictions of yield, solubility, and disorder matched the wet-lab outcome. This is a single, deliberately scoped case study, not a population-level benchmark; the two constructs differ in more than one feature, and biological activity was not assayed. Alongside the bottlenecks of this approach discussed here, used as a decision aid, prediction-guided construct and protocol design has the potential to remove costly iteration cycles of protein production campaigns.

Background. Scanning electron microscopy (SEM) is widely used to determine fibrin fiber structural properties such as fiber diameter and fiber length. However, conventional SEM preparation protocols are time-consuming and typically require conductive sputter coating. The coating process introduces an additional layer onto the sample surface and may influence measurements of nanoscale fiber structure. Furthermore, preparation of purified fibrinogen clots often follows protocols originally developed for plasma clots, resulting in unnecessary processing steps. Objective. To evaluate indium tin oxide (ITO) as a flat, conductive substrate for SEM imaging of fibrin fibers, investigate the effects of sputter coating on measured fiber diameter, and develop a simplified SEM preparation protocol for purified fibrinogen clots. Methods. Platelet-poor plasma clots and purified fibrinogen clots were formed on ITO substrates and imaged by SEM following 0 s, 45 s, or 90 s sputter coating. Fibrin fiber diameters were quantified and compared across coating conditions. For purified fibrinogen clots, an ITO-based simplified preparation protocol, in which clots were formed and imaged directly on the conductive ITO surface, was compared with a previously developed, standardized SEM protocol, in which clots were formed in microtube lids and subsequently transferred onto carbon tape for imaging. Results. Fiber diameter measurements were affected by sputter coating duration, with increasing coating time resulting in larger apparent fiber diameters. Plasma and purified fibrinogen clots exhibited distinct fiber diameter distributions and coating responses. For purified fibrinogen clots, the simplified ITO-based protocol produced fiber diameter measurements that were not significantly different from those obtained using the standardized lid-to-carbon-tape workflow when identical coating times were applied. Conclusions. ITO provides a practical conductive substrate for SEM imaging of fibrin fibers and enables substantial simplification of purified fibrinogen clot preparation. When coating conditions are matched, the simplified ITO-based protocol yields fiber diameter measurements comparable to those obtained using the previously standardized lid-to-carbon-tape workflow. These findings support the use of ITO as an alternative conductive imaging substrate and provide a simplified workflow for SEM analysis of purified fibrinogen clots. By reducing washing and transfer steps, this workflow may also provide a useful platform for future controlled studies of fibrin interactions with added proteins or other associated components.

A priori assessment of target proteins' druggability remains an unsolved problem in the field of drug development. The empirical approaches widely used to solve this problem demonstrate low efficiency. In this work, we investigated the factor of hydration of a representative set of 65 evolutionarily and structurally unrelated human enzymes in a water environment. This factor depends only on the structure of the proteins, and not on the physical and chemical properties of any potential ligands. The results show that, unlike the widely used approaches based on calculations of the accessible surface area (ASA), the content of low-entropy water molecules (LEW) in the active sites of human enzymes is systematically higher than that in other areas of their surface, including inactive cavities. Optimal criteria and a step-by-step procedure for identifying protein ligand binding sites are proposed. The proposed approach, based on the calculation of the LEW content in the first hydration layer of potentially interesting target proteins, makes it possible to evaluate their medicinal suitability even before the development of any ligands. The article also presents the results of a comparative analysis of experimental Raman spectroscopy data and the results of molecular dynamics simulations of water hydrogen bonds using three widely used water models (TIP3P, OPC3, and TIP5P) and standard algorithms for calculating hydrogen bond networks.

Lipid droplets (LDs) are cellular organelles responsible for lipid storage and metabolism. The mechanism of biogenesis of LDs involves phase separation of neutral lipids from the surrounding phospholipids, which generates oil lenses embedded in lipid bilayers, also known as nascent LDs. As nascent LDs grow, at some point they bud out of the bilayer, forming nearly spherical droplets. Nascent LDs have different propensity to bud, and it has been proposed that their shape provides information on such propensity; however, LD shape is difficult to determine experimentally. Here we studied the shape of lipid droplets using MD simulations at the coarse-grained level, and compared it to the predictions by an established theory. Our general system setup features an oil lens embedded into a flat, periodic bilayer. We found that the shape of simulated nascent LDs resembles a spherical cap (i.e., it has constant curvature over most of the surface), in excellent agreement with the theory, already for very small droplet sizes. The aspect ratio (height/radius) of nascent LDs increases with increasing LD volume, increasing membrane softness, and increasing surface tension between oil and water, also in agreement with theoretical predictions; however, it remains lower than 1 (i.e., the ratio for a sphere) for LDs of up to 40 nm in diameter. Fitting the simulated LD shapes with a theoretical shape equation suggests that a non-zero surface tension is present in both the monolayer and in the bilayer region. The existence of a relatively high surface tension in the bilayer region is confirmed by local stress calculations, and indicates that the periodic system setup does not reproduce the properties of nascent LDs in the endoplasmic reticulum, where the bilayer tension is two orders of magnitude lower. However, the simulations provide a microscopic view into the properties of droplet embedded vesicles.

Microscopes are essential tools for discoveries on a scale invisible to the unaided human eye. The development of immuno-fluorescence followed by molecular biology techniques and fluorescent fusion proteins have revolutionised the use of optical microscopy in bioscience. The quality of the data produced is dependent upon the sample, its preparation and the instrument used. However, instruments can degrade over time without easily visible changes to the produced images and, in turn, negatively impacts results. By testing instruments and doing comparisons between results over time and between different instruments, problems can be highlighted and corrective action can be taken. Using small fluorescent beads the point spread function (PSF) of the microscope can be recorded and the image resolution measured. Beads were prepared in a concentration matched to the field of view size and dried onto coverslips and mounted on slides. The beads were then imaged as 3D Z-stacks of sufficient size to fully enclose the PSF of the system. This data was uploaded to OMERO and processed using OMERO-metrics, an OMERO plugin developed for this purpose. This paper summarizes the development of workflows and protocols to enable this process, presents the results obtained and demonstrates the detection of significant instrument issues.

Glycosaminoglycans (GAGs) are linear, negatively charged, polysaccharides that mediate a wide variety of biologically critical interactions with proteins, underpinning growth factor signalling, extracellular matrix assembly and numerous disease processes. However, GAG-protein interactions remain under characterised, in part because of the lack of high-throughput tools to systematically profile binding across the GAG interactome. In this paper we present a novel Surface Plasmon Resonance-based array methodology utilising 16 commonly sourced GAG preparations (including chondroitin sulphate (CS), dermatan sulphate (DS), heparan sulphate, heparin, hyaluronan and keratan sulphate) allowing the specificity and affinity of GAG-binding proteins to be determined. As proof of principle, we have validated the array using four established GAG-binding proteins (antithrombin III, CD44, heavy chain 1 from inter--inhibitor and Slit2), generating data consistent with the known binding specificities and quantifying affinities for many of the interactions. The array also reveals previously unreported GAG interactions, including Slit2 binding to CS and DS, and CD44 binding to chondroitin sulphate E.

Accurate evaluation of extracellular matrix (ECM) mineralization at the nano-scale is essential for establishing relevant in vitro bone models. This is particularly important with the development and increased application of three-dimensional (3D) cell models for biological research. Transmission electron microscopy (TEM) allows to perform ultra-structural analysis of cells and ECM organization, but its application in in vitro bone models remains limited, due to the potential alteration or loss of the mineral phase during sample preparation. In this study, we compared two TEM sample preparation methods - the conventional chemical fixation and the anhydrous methods - to evaluate their ability to preserve the mineralized ECM in MC3T3-E1 cells cultured as monolayers and as alginate-encapsulated bone spheroids. Chemical fixation preserved cellular ultra-structure and collagen organization, allowing for detailed assessment of cells and ECM organization. Although mineral deposits were detected and their needle-like morphology assessed, characterization of more immature deposits was partially limited by the effects of uranyl acetate and the overall sample preparation process, which could lead to alteration or loss of less stable mineral phases. The anhydrous preparation method resulted in limited preservation of cellular and ECM morphology and did not allow reliable identification of mineral deposits. When applied to spheroids, the chemical fixation method preserved the 3D architecture, collagen-rich ECM and inner mineral deposits, confirming spheroids as a relevant model for bone studies. Overall, these results highlight the need for optimized sample preparation strategies that preserve both ultra-structure and mineral components for accurate nano-scale characterization of bone mineralization.

The growing number of approved nucleic acid therapeutics illustrates the potential to treat diseases by targeting their genetic blueprints in vivo. The 10-23 DNAzyme is capable of cleaving a wide range of target RNA with high selectivity. However, its poor performance in vivo restricts its therapeutic application as gene silencing agent. Studies on ribozymes have shown that the crowded environment in cells and associated effects can impact ribozyme folding and thermostability, resulting in a change in activity. This opens up the question whether DNAzymes are also affected by molecular crowding. Here, we investigate the functional and structural influence of molecular crowding conditions on the 10-23 DNAzyme. The stability and activity of a PrP-specific 10-23 DNAzyme were examined in presence of PEG, dextran, and osmolytes. Our results indicate that osmolytes decrease DNAzyme activity in a concentration-dependent manner, while certain PEG and dextran concentrations promote activity. To rationalize our observations, we studied the cosolutes effect on physicochemical solution properties and the structure of the DNAzyme:RNA complex using FCS and SAXS. The data reveal that enhanced activity is observed under conditions where a combination of physiochemical properties matches an optimum that seems to be dependent on the metal ion cofactor. Structural influence under such conditions is indicated less. We propose that a certain degree of molecular crowding is required to favor a state, which allows for higher catalytic turnover. In addition, we show that the requirement for magnesium and manganese as a cofactor remains unchanged under the conditions applied. Our work contributes to a better understanding of how the cellular environment affects DNAzyme structure and function.

BackgroundThe physical properties of leukocytes, such as cell size and stiffness, are critical for their circulation in microcapillary networks where rapid shape changes are required to squeeze through vascular constrictions. Alterations in the cells physical phenotype can promote venous thromboembolism (VTE), a common cause of death in cancer patients receiving chemotherapy. While biochemical VTE predictors are well studied, physical properties of blood cells receive less attention. MethodsUsing real-time deformability cytometry (RT-DC), we monitored for the first time the physical phenotype of leukocytes in a longitudinal study of a breast cancer patient treated with epirubicin/cyclophosphamide (EC) and paclitaxel (Pax). ResultsThe leukocyte counts extracted from RT-DC were in good agreement with standard clinical leukograms and EC had no immediate effect on leukocyte properties. However, Pax caused a significant softening of granulo/monocytes and a stiffening of lymphocytes immediately after administration. Leukocyte size was constant throughout the therapy, but we observed an overall increase in leukocyte stiffness, which was restored to normal values 45 weeks post treatment. ConclusionTaken together, our data reveal chemotherapy-induced specific alterations of leukocyte stiffness potentially critical for microcirculation. Thus, RT-DC measurements can add important, yet currently not available information to VTE prediction in cancer patients.

Chemical imaging is a powerful branch of modern microscopy encumbered by a lack of flexible, high-throughput analysis tools. Bespoke analytical pipelines typically perform ratiometric analysis on two layers in a multispectral image to describe the relative composition of molecules in a sample. This strategy has been implemented across fields, spanning histopathology, cell biology, environmental science, and materials science. The commercialization of chemical imaging microscopes has facilitated the collection of large multispectral datasets, necessitating accessible ways to process them. This paper describes Multispectral Analysis Graphical User Interface (msaGUI), a desktop graphical user interface to analyze individual and batch datasets of multispectral images. Data is loaded as CSV, TSV, or TIFFs and processed through a user-defined sequence of modular image operations that can be flexibly combined, e.g. to reduce spectral crosstalk or background noise. After analysis, data is visualized as exportable images, histograms, and statistics. To yield publication-quality figures, outputted images are fully customizable. Written in Python with open-source libraries, the msaGUI program is packaged into an executable for Windows and Mac for a fully no-code application. Other operating systems are supported via the Python source code. In summary, msaGUI provides a rapid and user-friendly solution for analyzing and visualizing multispectral data.

The glycocalyx of red blood cells (RBCs), a negatively charged surface layer rich in sialic acid residues, plays a crucial role in modulating RBC aggregation. In pathological conditions such as diabetes and sepsis, glycocalyx degradation is often observed along with abnormal RBC aggregation. However, the mechanistic relationship between these phenomena remains poorly defined. In this study, we investigate the effects of enzymatic glycocalyx degradation on RBC aggregation under physiologically relevant flow conditions. Using neuraminidase from Clostridium perfringens (C. welchii) at varying concentrations, we selectively removed sialic acid residues from the RBC glycocalyx, simulating different levels of desialylation observed in health and disease. Confocal microscopy confirmed the dose-dependent depletion of membrane sialic acid, while microfluidic experiments revealed a significant increase in both the size and stability of the RBC aggregates after enzymatic treatment. Our findings suggest that glycocalyx integrity is a crucial biophysical determinant of RBC aggregation, likely influencing both electrostatic repulsion and hydrodynamic forces. This study provides new insights into how the enzymatic modification of the glycocalyx contributes to pathological hemorheology and may inform future strategies for the diagnosis or treatment of vascular diseases.

The ParABS system orchestrates chromosome segregation in many bacterial species. The centromere-like parS sites serve as nucleation points for the initial binding of the ParB protein. Subsequent diffusion on adjacent, non-specific DNA regions (spreading) in the presence of CTP and binding of more ParB molecules along with DNA looping via ParB-ParB interactions bring distal parts of the chromosome into proximity. ParB interaction with the ParA-ATPase motor protein, then, drives genomic segregation. It has been shown that in some bacterial species, the ParB-parS complex undergoes phase separation into a condensate. However, the physico-chemical properties of such condensates and their response to forces, such as those they may face in the cell, have not yet been characterized. Performing turbidity measurements in the presence of CTP and various concentrations of DNA and physiologically relevant mono and divalent salt It was shown that Mg2+ facilitates, while K+ concentrations higher than [~]20 mM disfavors, condensate formation. Microrheology measurements showed that condensates of ParB and DNA including parS sites (ParB-parS DNA) in the presence of CTP, are viscoelastic with a viscosity at Troom of [~]5 Pa{middle dot}s and able to quickly respond to deformations with a network relaxation time of 0.1 s. Additionally, fluorescence combined with force spectroscopy showed that mechanical disruption of ParB-DNA condensates in the presence of CTP requires [~] 5-7.5 pN of tension in the DNA, which is lower than the force required to stall a molecular motor such as RNA polymerase, but higher than the force required for the relocation of chromosomes and plasmids during segregation. These results support the idea that ParB-parS condensates dynamically rearrange at the molecular level while maintaining the cohesion necessary to sustain the drag force of segregation without interfering with genomic transactions. This physical mechanism could be the basis for the critical role of ParB-parS condensates in organizing and partitioning bacterial chromosomes. Statement of SignificanceCondensates formed by liquid-liquid phase separation enable cellular compartmentalization without the energy-costly production of a membrane enclosure and have been implicated in a wide array of cellular functions. Here, we analyze their chemo-physical properties in relation to their activity in bacterial chromosome segregation using the C. crescentus ParB protein and DNA containing parS specific binding sites. We find that the cohesion of condensates of ParB parS-containing DNA confers the mechanical stability necessary to pull genetic material towards the cell pole without interfering with genomic functions. We propose a mechanism to explain the function of ParB-parS DNA in chromosomal segregation.

There is a vast literature on the voltage gating of ion channels, with a fairly large fraction concerned with potassium channels, especially of the KV1 family, including Shaker. Experimental evidence derived from protein structure has been interpreted to give gating mechanisms that largely disregard water. We propose that the K+ ion, in order to pass through the gating region and enter the cavity pore, must be largely dehydrated. Competitive interactions of each single hydration shell water at the gate, with counterions, protein, or other water molecules, can remove one water at a time. There are several such interactions for the ion hydration shell; for the ion to pass through the gating region, there must be enough such interactions to leave the ion with at most two hydrating water molecules, in which case the gate is open. Protein conformational changes are secondary, small, and mostly unimportant. The hypothesis has a second part: protons, previously shown to be candidate carriers of the gating current (Kariev and Green, JPC B, 2019, Membranes, 2022, 2024) are capable of reaching the gate; adding four protons to the gate prevents dehydration, leaving the ion with at least three hydrating water molecules, enough to block passage. Quantum calculations presented here support the dehydration part of the hypothesis; they also mostly support the second part, concerning the protons, but further work will be required to fully confirm this. The hypothesis explains the experimental finding that the P475D mutant is essentially constitutively open, while the P475S mutant, with a wider gate opening, is closed at all relevant potentials; the computations presented here show the mechanism for this in detail, further confirming the first part of the hypothesis, and largely but not completely confirming the second part, concerning protons, while showing where further work is needed. This mechanism can also qualitatively account for flicker noise and fluctuations, and their consequences.