Counter Anion Type Influences the Glass Transition Temperature of Polyelectrolyte Complexes.

Salt acts as a plasticizer in polyelectrolyte complexes (PECs), which impacts the physical, thermal, and mechanical properties, thus having implications in applications, such as drug delivery, energy storage, and smart coatings. Added salt disrupts polycation-polyanion intrinsic ion pairs, lowering a hydrated PEC's glass transition temperature (Tg). However, the relative influence of counterion type on the PEC's Tg is not well understood. Here, the effect of anion type (NaCl, NaBr, NaNO3, and NaI) on the Tg of solid-like, hydrated PECs composed of poly(diallydimethylammonium) (PDADMA)-poly(styrenesulfonate) (PSS) is investigated. With increasing the chaotropic nature of the salt anion, the Tg decreases. The relative differences are attributed to the doping level, the amount of bound water, the mobility of water molecules within the PECs, and the strength of interactions between the PEs. For all studied salt concentrations and salt types, the Tg followed the scaling of -1/Tg ≈ ln([IP]/[H2O]), in which [IP]/[H2O] is the ratio of intrinsic pairs to water. The scaling estimates that about 7 to 17% of the intrinsic ion pairs should be weakened for the PEC to partake in a glass transition. Put together, this study highlights that the Tg in PECs is impacted by the salt anion, but the mechanism of the glass transition remains unchanged.

Ion-Specific Effects on Ion and Polyelectrolyte Solvation.

Ion-specific effects on aqueous solvation of monovalent counter ions, Na + ${^+ }$ , K + ${^+ }$ , Cl - ${^- }$ , and Br - ${^- }$ , and two model polyelectrolytes (PEs), poly(styrene sulfonate) (PSS) and poly(diallyldimethylammonium) (PDADMA) were here studied with ab initio molecular dynamics (AIMD) and classical molecular dynamics (MD) simulations based on the OPLS-aa force-field which is an empirical fixed point-charge force-field. Ion-specific binding to the PE charge groups was also characterized. Both computational methods predict similar response for the solvation of the PEs but differ notably in description of ion solvation. Notably, AIMD captures the experimentally observed differences in Cl - ${^- }$ and Br - ${^- }$ anion solvation and binding with the PEs, while the classical MD simulations fail to differentiate the ion species response. Furthermore, the findings show that combining AIMD with the computationally less costly classical MD simulations allows benefiting from both the increased accuracy and statistics reach.

Accelerated Engineering of ELP-Based Materials through Hybrid Biomimetic-De Novo Predictive Molecular Design.

Efforts to engineer high-performance protein-based materials inspired by nature have mostly focused on altering naturally occurring sequences to confer the desired functionalities, whereas de novo design lags significantly behind and calls for unconventional innovative approaches. Here, using partially disordered elastin-like polypeptides (ELPs) as initial building blocks this work shows that de novo engineering of protein materials can be accelerated through hybrid biomimetic design, which this work achieves by integrating computational modeling, deep neural network, and recombinant DNA technology. This generalizable approach involves incorporating a series of de novo-designed sequences with α-helical conformation and genetically encoding them into biologically inspired intrinsically disordered repeating motifs. The new ELP variants maintain structural conformation and showed tunable supramolecular self-assembly out of thermal equilibrium with phase behavior in vitro. This work illustrates the effective translation of the predicted molecular designs in structural and functional materials. The proposed methodology can be applied to a broad range of partially disordered biomacromolecules and potentially pave the way toward the discovery of novel structural proteins.

Solid-Liquid-Solution Phases in Poly(diallyldimethylammonium)/Poly(acrylic acid) Polyelectrolyte Complexes at Varying Temperatures.

The coacervation and complexation of oppositely charged polyelectrolytes are dependent on numerous environmental and preparatory factors, but temperature is often overlooked. Temperature effects remain unclear because the temperature dependence of both the dielectric constant and polymer-solvent interaction parameter can yield lower and/or upper critical solution phase behaviors for PECs. Further, secondary interactions, such as hydrogen bonding, can affect the temperature response of a PEC. That is, mixtures of oppositely charged polyelectrolytes can exhibit phase separation upon lowering and/or increasing the mixture's temperature. Here, the phase behavior of poly(diallylmethylammonium)/poly(acrylic acid) (PDADMA/PAA) complexes under varying KBr ionic strengths, mixing ratios, and temperatures at a fixed pH (in which PAA hydrogen bonding can occur) is examined. At room temperature, the PDADMA/PAA PECs exhibit four different phase states: precipitate, coexisting precipitate and coacervate, solid-like gel, and coacervate. Variable-temperature optical microscopy reveals the upper critical solution temperature (UCST) at which each phase transitioned to a solution state. Interestingly, the UCST value is highly dependent on the original phase of the PEC, in which solid-like precipitates exhibit higher UCST values. Large-scale all-atom molecular dynamics (MD) simulations support that precipitates exhibit kinetic trapping, which may contribute to the higher UCST values observed in the experiment. Taken together, this study highlights the significance of temperature on the phase behavior of PECs, which may play a larger role in stimuli-responsive materials, membraneless organelles, and separations applications.

Exploration of Chemical Diversity in Intercellular Quorum Sensing Signalling Systems in Prokaryotes.

Quorum sensing (QS) serves as a vital means of intercellular signalling in a variety of prokaryotes, which enables single cells to act in multicellular configurations. The potential to control community-wide responses has also sparked numerous recent biotechnological innovations. However, our capacity to utilize intercellular communication is hindered due to a scarcity of complementary signalling systems and a restricted comprehension of interconnections between these systems caused by variations in their dynamic range. In this study, we utilize uniform manifold approximation and projection and extended-connectivity fingerprints to explore the available chemical space of QS signalling molecules. We investigate and experimentally characterize a set of closely related QS signalling ligands, consisting of N-acyl homoserine lactones and the aryl homoserine lactone p-coumaroyl, as well as a set of more widely diverging QS ligands, consisting of photopyrones, dialkylresorcinols, 3,5-dimethylpyrazin-2-ol and autoinducer-2, and define their performance. We report on a set of six signal- and promoter-orthogonal intercellular QS signalling systems, significantly expanding the toolkit for engineering community-wide behaviour. Furthermore, we demonstrate that ligand diversity can serve as a statistically significant tool to predict much more complicated ligand-receptor interactions. This approach highlights the potential of dimensionality reduction to explore chemical diversity in microbial dynamics.

Mechanisms of Fibroblast Growth Factor 21 Adsorption on Macroion Layers: Molecular Dynamics Modeling and Kinetic Measurements.

Molecular dynamic modeling and various experimental techniques, including multi-angle dynamic light scattering (MADLS), streaming potential, optical waveguide light spectroscopy (OWLS), quartz crystal microbalance with dissipation (QCM), and atomic force microscopy (AFM), were applied to determine the basic physicochemical parameters of fibroblast growth factor 21 in electrolyte solutions. The protein size and shape, cross-section area, dependence of the nominal charge on pH, and isoelectric point of 5.3 were acquired. These data enabled the interpretation of the adsorption kinetics of FGF 21 on bare and macrocation-covered silica investigated by OWLS and QCM. It was confirmed that the protein molecules irreversibly adsorbed on the latter substrate, forming layers with controlled coverage up to 0.8 mg m-2, while their adsorption on bare silica was much smaller. The viability of two cell lines, CHO-K1 and L-929, on both bare and macrocation/FGF 21-covered substrates was also determined. It is postulated that the acquired results can serve as useful reference systems for designing complexes that can extend the half-life of FGF 21 in its active state.

A Perspective on the Glass Transition and the Dynamics of Polyelectrolyte Multilayers and Complexes.

Polyelectrolyte multilayers (PEMs) or polyelectrolyte complexes (PECs), formed by layer-by-layer assembly or the mixing of oppositely charged polyelectrolytes (PEs) in aqueous solution, respectively, have potential applications in health, energy, and the environment. PEMs and PECs are very tunable because their structure and properties are influenced by factors such as pH, ionic strength, salt type, humidity, and temperature. Therefore, it is increasingly important to understand how these factors affect PECs and PEMs on a molecular level. In this Feature Article, we summarize our contributions to the field in the development of approaches to quantify the swelling, thermal properties, and dynamic mechanical properties of PEMs and PECs. First, the role of water as a plasticizer and in the glass-transition temperature (Tg) in both strong poly(diallyldimethylammonium)/poly(sodium 4-styrenesulfonate) (PDADMA/PSS) and weak poly(allylamine hydrochloride)/poly(acrylic acid) (PAH/PAA) systems is presented. Then, factors influencing the dynamics of PECs and PEMs are discussed. We also reflect on the swelling of PEMs in response to different salts and solvent additives. Last, the nature of water's microenvironment in PEMs/PECs is discussed. A special emphasis is placed on experimental techniques, along with molecular simulations. Taken together, this review presents an outlook and offers recommendations for future research directions, such as studying the additional effects of hydrogen-bonding hydrophobic interactions.

Nanoscale insights into the local structural rearrangements of amyloid-ß induced by bexarotene.

A better understanding of the abnormal protein aggregation and the effect of anti-aggregation agents on the fibrillation pathways and the secondary structure of aggregates can determine strategies for the early treatment of dementia. Herein, we present a combination of experimental and theoretical studies providing new insights into the influence of the anti-aggregation drug bexarotene on the secondary structure of individual amyloid-ß aggregates and its primary aggregation. The molecular rearrangements and the spatial distribution of ß-sheets within individual aggregates were monitored at the nanoscale with infrared nanospectroscopy. We observed that bexarotene limits the parallel ß-sheets formation, known to be highly abundant in fibrils at later phases of the amyloid-ß aggregation composed of in-register cross-ß structure. Moreover, we applied molecular dynamics to provide molecular-level insights into the investigated system. Both theoretical and experimental results revealed that bexarotene slows down the protein aggregation process via steric effects, largely prohibiting the antiparallel to parallel ß-sheet rearrangement. We also found that bexarotene interacts not only via the single hydrogen bond formation with the peptide backbone but also with the amino acid side residue via a hydrophobic effect. The studied model of the drug-amyloid-ß interaction contributes to a better understanding of the inhibition mechanism of the amyloid-ß aggregation by the small molecule drugs. However, our nanoscale findings need to meet in vivo research requiring different analytical approaches.

pH dependence of the assembly mechanism and properties of poly(L-lysine) and poly(L-glutamic acid) complexes.

We show by extensive experimental characterization combined with molecular simulations that pH has a major impact on the assembly mechanism and properties of poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) complexes. A combination of dynamic light scattering (DLS) and laser Doppler velocimetry (LDV) is used to assess the complexation, charge state, and other physical characteristics of the complexes, isothermal titration calorimetry (ITC) is used to examine the complexation thermodynamics, and circular dichroism (CD) is used to extract the polypeptides' secondary structure. For enhanced analysis and interpretation of the data, analytical ultracentrifugation (AUC) is used to define the precise molecular weights and solution association of the peptides. Molecular dynamics simulations reveal the associated intra- and intermolecular binding changes in terms of intrinsic vs. extrinsic charge compensation, the role of hydrogen bonding, and secondary structure changes, aiding in the interpretation of the experimental data. We combine the data to reveal the pH dependency of PLL/PGA complexation and the associated molecular level mechanisms. This work shows that not only pH provides a means to control complex formation but also that the associated changes in the secondary structure and binding conformation can be systematically used to control materials assembly. This gives access to rational design of peptide materials via pH control.

SARS-CoV-2 Spike Protein (RBD) Subunit Adsorption at Abiotic Surfaces and Corona Formation at Polymer Particles.

The adsorption kinetics of the SARS-CoV-2 spike protein subunit with the receptor binding domain at abiotic surfaces was investigated. A combination of sensitive methods was used such as atomic force microscopy yielding a molecular resolution, a quartz microbalance, and optical waveguide lightmode spectroscopy. The two latter methods yielded in situ information about the protein adsorption kinetics under flow conditions. It was established that at pH 3.5-4 the protein adsorbed on mica and silica surfaces in the form of compact quasi-spherical aggregates with an average size of 14 nm. The maximum coverage of the layers was equal to 3 and 1 mg m-2 at pH 4 and 7.4, respectively. The experimental data were successfully interpreted in terms of theoretical results derived from modeling. The experiments performed for flat substrates were complemented by investigations of the protein corona formation at polymer particles carried out using in situ laser Doppler velocimetry technique. In this way, the zeta potential of the protein layers was acquired as a function of the coverage. Applying the electrokinetic model, these primary data were converted to the dependence of the subunit zeta potential on pH. It was shown that a complete acid-base characteristic of the layer can be acquired only using nanomolar quantities of the protein.

Quantification of Water-Ion Pair Interactions in Polyelectrolyte Multilayers Using a Quartz Crystal Microbalance Method.

Water existing within thin polyelectrolyte multilayer (PEM) films has significant influence on their physical, chemical, and thermal properties, having implications for applications including energy storage, smart coatings, and biomedical systems. Ionic strength, salt type, and terminating layer are known to influence PEM swelling. However, knowledge of water's microenvironment within a PEM, whether that water is affiliated with intrinsic or extrinsic ion pairs, remains lacking. Here, we examine the influence of both assembly and post-assembly conditions on the water-ion pair interactions of poly(styrene sulfonate)/poly(diallyldimethylammonium) (PSS/PDADMA) PEMs in NaCl and KBr. This is accomplished by developing a methodology in which quartz crystal microbalance with dissipation monitoring is applied to estimate the number of water molecules affiliated with an ion pair (i), as well as the hydration coefficient, πsalt H2O. PSS/PDADMA PEMs are assembled in varying ionic strengths of either NaCl and KBr and then exposed post-assembly to increasing ionic strengths of matching salt type. A linear relationship between the total amount of water per intrinsic ion pair and the post-assembly salt concentration was obtained at post-assembly salt concentrations >0.5 M, yielding estimates for both i and πsalt H2O. We observe higher values of i and πsalt H2O in KBr-assembled PEMs due to KBr being more effective in doping the assembly because of KBr's more chaotropic nature as compared to NaCl. Lastly, when PSS is the terminating layer, i decreases in value due to PSS's hydrophobic nature. Classical and ab initio molecular dynamics provide a microstructural view as to how NaCl and KBr interact with individual polyelectrolytes and the involved water shells. Put together, this study provides further insight into the understanding of existing water microenvironments in PEMs and the effects of both assembly and post-assembly conditions.

Random sequential adsorption: An efficient tool for investigating the deposition of macromolecules and colloidal particles.

Random Sequential Adsorption (RSA) is one of the most efficient theoretical models used to investigate adsorption of macromolecules and particles, with a long-standing tradition in the field of colloid and interface science. In the first part of this paper, we demonstrate how the RSA model can be applied to interpret the experimental data and extract information about the density of the adsorption monolayer, the kinetics of its growth, and microstructural properties such as pair-correlation function and monolayer roughness. We briefly summarized the most important generalizations of the RSA model for monolayers and reviewed its extensions considering, e.g., various particle shapes, the introduction of electrostatic interaction, or adsorption on non-uniform substrates. We thoroughly scrutinized the extended RSA model developed for bilayer and multilayer formation. We collected the mean saturated packing fractions of various two- and three-dimensional objects and provided the most accurate result for two-dimensional disk packing. In the second part of this paper, we summarize various numerical algorithms and techniques that allow one to effectively implement RSA algorithms. We describe efficient methods for detecting intersections of various shapes and techniques enabling generation of strictly saturated RSA packings built of a wide range of different shapes. We hinted at how an inherently sequential RSA scheme can be parallelized. Finally, we critically discuss the limitations of the model and possible directions for future studies.

Effect of Ethanol and Urea as Solvent Additives on PSS-PDADMA Polyelectrolyte Complexation.

The effect of urea and ethanol additives on aqueous solutions of poly(styrenesulfonate) (PSS), poly(diallyldimethylammonium) (PDADMA), and their complexation interactions are examined here via molecular dynamics simulations, interconnected laser Doppler velocimetry, and quartz crystal microbalance with dissipation. It is found that urea and ethanol have significant, yet opposite influences on PSS and PDADMA solvation and interactions. Notably, ethanol is systematically depleted from solvating the charge groups but condenses at the hydrophobic backbone of PSS. As a consequence of the poorer solvation environment for the ionic groups, ethanol significantly increases the extent of counterion condensation. On the other hand, urea readily solvates both polyelectrolytes and replaces water in solvation. For PSS, urea causes disruption of the hydrogen bonding of the PSS headgroup with water. In PSS-PDADMA complexation, these differences influence changes in the binding configurations relative to the case of pure water. Specifically, added ethanol leads to loosening of the complex caused by the enhancement of counterion condensation; added urea pushes polyelectrolyte chains further apart because of the formation of a persistent solvation shell. In total, we find that the effects of urea and ethanol rise from changes in the microscopic-level solvation environment and conformation resulting from solvating water being replaced by the additive. The differences cannot be explained purely via considering relative permittivity and continuum level electrostatic screening. Taken together, the findings could bear significance in tuning polyelectrolyte materials' mechanical and swelling characteristics via solution additives.

Poly-L-Arginine Molecule Properties in Simple Electrolytes: Molecular Dynamic Modeling and Experiments.

Physicochemical properties of poly-L-arginine (P-Arg) molecules in NaCl solutions were determined by molecular dynamics (MD) modeling and various experimental techniques. Primarily, the molecule conformations, the monomer length and the chain diameter were theoretically calculated. These results were used to interpret experimental data, which comprised the molecule secondary structure, the diffusion coefficient, the hydrodynamic diameter and the electrophoretic mobility determined at various ionic strengths and pHs. Using these data, the electrokinetic charge and the effective ionization degree of P-Arg molecules were determined. In addition, the dynamic viscosity measurements for dilute P-Arg solutions enabledto determine the molecule intrinsic viscosity, which was equal to 500 and 90 for ionic strength of 10-5 and 0.15 M, respectively. This confirmed that P-Arg molecules assumed extended conformations and approached the slender body limit at the low range of ionic strength. The experimental data were also used to determine the molecule length and the chain diameter, which agreed with theoretical predictions. Exploiting these results, a robust method for determining the molar mass of P-Arg samples, the hydrodynamic diameter, the radius of gyration and the sedimentation coefficient was proposed.

SARS-CoV-2 virion physicochemical characteristics pertinent to abiotic substrate attachment.

The structure, size, and main physicochemical characteristics of the SARS-CoV-2 virion with the spike transmembrane protein corona were discussed. Using these data, diffusion coefficients of the virion in aqueous media and in air were calculated. The structure and dimensions of the spike protein derived from molecular dynamic modeling and thorough cryo-electron microscopy measurements were also analyzed. The charge distribution over the molecule was calculated and shown to be largely heterogeneous. Although the stalk part is negatively charged, the top part of the spike molecule, especially the receptor binding domain, remains positively charged for a broad range of pH. It is underlined that such a charge distribution promotes the spike corona stability and enhances the virion attachment to receptors and surfaces, mostly negatively charged. The review is completed by the analysis of experimental data pertinent to the spike protein adsorption at abiotic surfaces comprising nanoparticle carrier particles. It is argued that these theoretical and experimental data can be used for developing quantitative models of virus attachment to surfaces, facilitating adequate analysis of future experimental results.

Synergistic Effect of Binary Surfactant Mixtures in Two-Phase and Three-Phase Systems.

Cationic alkyltrimethylammonium bromides (CnTAB, with n = 8, 12, 16, 18) and their mixtures with n-octanol as a nonionic surfactant were chosen as a model system to study the synergistic effect on foamability (two-phase system) and floatability (three-phase system) of quartz in the presence of binary mixtures of ionic/nonionic surfactants. The foam height of one-component solutions and binary mixtures and floatability of quartz particles were characterized as a function of the surfactant concentration and the number of carbons (n) in the alkyl chain of CnTAB. The experimental results of foamability and floatability measurements in one-component and mixed solutions revealed the synergistic effect, causing a significant enhancement in the foam height and recovery of quartz. In the presence of n-octanol, the height of foam increased remarkably for all CnTAB solutions studied, and this effect, whose magnitude depended on the CnTAB hydrophobic tail length, could not be justified by a simple increase in total surfactant concentration. A similar picture was obtained in the case of flotation response. The mechanism of synergistic effect observed in mixed CnTAB/n-octanol solutions was proposed. The discussion was supported by molecular dynamics simulations, and the probable mechanism responsible for synergism was discussed. In addition, an analysis allowing accurate determination of the concentration regimes, where the synergistic effect can be expected, was given. It was shown that for the two-phase system, the n-octanol molecule preadsorption at the liquid/gas interface causes an increase in CnTAB adsorption coverage over the level expected from its equilibrium value in the one-component solution. In the case of the three-phase system, the synergistic effect was related to the ionic surfactants serving as an anchor layer for n-octanol, which, in water/n-octanol solution (one-component system), do not adsorb on the surface of quartz.

Self-Assembly of Silk-like Protein into Nanoscale Bicontinuous Networks under Phase-Separation Conditions.

Liquid-liquid phase separation of biomacromolecules is crucial in various inter- and extracellular biological functions. This includes formation of condensates to control, e.g., biochemical reactions and structural assembly. The same phenomenon is also found to be critically important in protein-based high-performance biological materials. Here, we use a well-characterized model triblock protein system to demonstrate the molecular level formation mechanism and structure of its condensate. Large-scale molecular modeling supported by analytical ultracentrifuge characterization combined with our earlier high magnification precision cryo-SEM microscopy imaging leads to deducing that the condensate has a bicontinuous network structure. The bicontinuous network rises from the proteins having a combination of sites with stronger mutual attraction and multiple weakly attractive regions connected by flexible, multiconfigurational linker regions. These attractive sites and regions behave as stickers of varying adhesion strength. For the examined model triblock protein construct, the ß-sheet-rich end units are the stronger stickers, while additional weaker stickers, contributing to the condensation affinity, rise from spring-like connections in the flexible middle region of the protein. The combination of stronger and weaker sticker-like connections and the flexible regions between the stickers result in a versatile, liquid-like, self-healing structure. This structure also explains the high flexibility, easy deformability, and diffusion of the proteins, decreasing only 10-100 times in the bicontinuous network formed in the condensate phase in comparison to dilute protein solution. The here demonstrated structure and condensation mechanism of a model triblock protein construct via a combination of the stronger binding regions and the weaker, flexible sacrificial-bond-like network as well as its generalizability via polymer sticker models provide means to not only understand intracellular organization, regulation, and cellular function but also to identify direct control factors for and to enable engineering improved protein and polymer constructs to enhance control of advanced fiber materials, smart liquid biointerfaces, or self-healing matrices for pharmaceutics or bioengineering materials.

Adsorption kinetic of myoglobin on mica and silica - Role of electrostatic interactions.

Adsorption kinetics of myoglobin molecules on mica and silica was studied using the atomic force microscopy (AFM), the colloid enhancement and the quartz microbalance (QCM) methods. Measurements were carried out for the NaCl concentration of 0.01 and 0.15 M as a function of pH comprising pH 7.4 stabilized by the PBS buffer. The electrophoretic mobility measurements enabled to derive the molecules zeta potential as a function of pH. The isoelectric point appearing at pH 5, is lower than that predicted from the theoretical calculations of the nominal dissociation charge. The AFM investigations confirmed that myoglobin molecules irreversibly adsorb at pH 3.5 yielding well-defined layers of single molecules. These layers were characterized using the colloid enhancement method involving polymer microparticles for pH range 3-9. The microparticle deposition kinetics was adequately interpreted in terms of a hybrid random sequential adsorption model. It is confirmed that the myoglobin layers exhibit a negligible zeta potential at pH equal to 5 in accordance with the electrophoretic mobility measurements. Analogous adsorption kinetic measurements were performed for the silica substrate using QCM and AFM. It is observed that myoglobin molecules irreversibly adsorb at pH 3.5 forming stable layers of single molecules. On the other hand, its adsorption kinetics at larger pHs was much slower exhibiting a poorly defined maximum coverage. This was attributed to aggregation of the myoglobin solutions due to their vanishing charge. The kinetic QCM runs were adequately interpreted in terms of a theoretical model combining the Smoluchowski aggregation theory with the convective diffusion mass transfer theory.

Myoglobin molecule charging in electrolyte solutions.

All atom molecular dynamic modeling was applied in order to determine water molecule and electrolyte ion concentration profiles around and inside the myoglobin molecule at various pH values. Significant penetration of counter ions into the molecule was confirmed. The electric potential distribution within and outside the molecule was quantitatively described using the non-linear Poisson-Boltzmann (PB) approach. Using this model, calculations were performed, yielding the surface and zeta potential for various physicochemical parameters, comprising pH, the electric permittivity, the ion penetration depth and the protein volume fraction (crowding effect). The theoretical results were used for the interpretation of experimental data acquired under different ionic strengths and temperatures by electrophoretic mobility measurements. It is confirmed that the experimental data are adequately reflected for acidic pH values by the non-linear PB model where the nominal molecule charge was calculated from the H++ model. The deviations occurring for larger pH values were accounted for by considering additional non-electrostatic interactions stemming from the van der Waals and ion-induced dipole forces. In this way, it is both experimentally and theoretically confirmed that the effective charge of the myoglobin molecule in electrolyte solutions is considerably smaller than the nominal, structure-based, predicted charge. As a result, under physiological conditions prevailing, e.g. in skeletal muscles, the effective charge of the myoglobin molecule should practically vanish. One can expect that the approach developed in this work can be applied for predicting charging mechanisms of other protein molecules characterized by an analogous charge vs. pH characteristic, e.g., the SARS-CoV-2 virus spike proteins, and for soft particles with pH responsive characteristics.

Analyzing the weak dimerization of a cellulose binding module by analytical ultracentrifugation.

Cellulose binding modules (CBMs) are found widely in different proteins that act on cellulose. Because they allow a very easy way of binding recombinant proteins to cellulose, they have become widespread in many biotechnological applications involving cellulose. One commonly used variant is the CBMCipA from Clostridium thermocellum. Here we studied the oligomerization behavior of CBMCipA, as such solution association may have an impact on its use. As the principal approach, we used sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. To enhance our understanding of the possible interactions, we used molecular dynamics simulations. By analysis of the sedimentation velocity data by a discrete model genetic algorithm and by building a binding isotherm based on weight average sedimentation coefficient and by global fitting of sedimentation equilibrium data we found that the CBMCipA shows a weak dimerization interaction with a dissociation constant KD of 90 ± 30 µM. As the KD of CBMCipA binding to cellulose is below 1 µM, we conclude that the dimerization is unlikely to affect cellulose binding. However, at high concentrations used in some applications of the CBMCipA, its dimerization is likely to have a marked effect on its solution behavior.