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The structural properties of the salivary protein statherin upon adsorption have been examined using a coarse-grained model and Monte Carlo simulation. A simple model system with focus on electrostatic interactions and short-ranged attractions among the uncharged amino acids has been used. To mimic hydrophobically modified surfaces, an extra short-ranged interaction was implemented between the amino acids and the surface. It has been shown that the adsorption and the thickness of the adsorbed layer are determined by (i) the affinity for the surface, i.e., denser layer with an extrashort-ranged potential, and (ii) the distribution of the charges along the chain. If all the amino acids have a high affinity for the surface, the protein adsorbs in a train conformation, if the surface is negatively charged the protein adsorbs in a tail-train conformation, whereas if the surface is positively charged the protein adsorbs in a loop conformation. The latter gives rise to a more confined adsorbed layer. [ABSTRACT FROM AUTHOR]

Polyion conformation and the distribution of small ions near the polyion have been investigated using Monte Carlo simulations. The systems of interest contained one polyion and its monovalent counterions, and variable amount of a 3:1 salt. With monovalent counterions only, the polyion is strongly extended. As salt is added, the polyion folds, and the most compact and spherical-like structure appears at a three-fold excess of the trivalent counterions. The polyion exerts a strong influence on the nearest-neighbor distance among the trivalent ions, an effect being relevant for energy transfer reactions. © 2003 American Institute of Physics. [ABSTRACT FROM AUTHOR]

It is generally known that, unlike structured proteins, intrinsically disordered proteins, IDPs, exhibit various structures and conformers, the so-called conformational ensemble, CoE. This study aims to better understand the conformers that make up the IDP ensemble by decomposing the CoE into groups separated by their radius of gyration, R g . A common approach to studying CoE for IDPs is to use low-resolution techniques, such as small-angle scattering, and combine those with computer simulations on different length scales. Herein, the well-studied antimicrobial saliva protein histatin 5 was utilized as a model peptide for an IDP; the average intensity curves were obtained from small-angle X-ray scattering; and compared with fully atomistic, explicit water, molecular dynamics simulations; then, the intensity curve was decomposed with respect to the different R g values; and their secondary structure propensities were investigated. We foresee that this approach can provide important information on the CoE and the individual conformers within; in that case, it will serve as an additional tool for understanding the IDP structure-function relationship on a more detailed level.

Cushioned lipid bilayers are structures consisting of a lipid bilayer supported on a solid substrate with an intervening layer of soft material. They offer possibilities for studying the behavior and interactions of biological membranes more accurately under physiological conditions. In this work, we continue our studies of cushion formation induced by histatin 5 ( 24 Hst5), focusing on the effect of the length of the peptide chain. 24 Hst5 is a short, positively charged, intrinsically disordered saliva peptide, and here, both a shorter ( 14 Hst5) and a longer ( 48 Hst5) peptide variant were evaluated. Experimental surface active techniques were combined with coarse-grained Monte Carlo simulations to obtain information about these peptides. Results show that at 10 mM NaCl, both the shorter and the longer peptide variants behave like 24 Hst5 and a cushion below the bilayer is formed. At 150 mM NaCl, however, no interaction is observed for 24 Hst5. On the contrary, a cushion is formed both in the case of 14 Hst5 and 48 Hst5, and in the latter, an additional thick, diffuse, and highly hydrated layer of peptide and lipid molecules is formed, on top of the bilayer. Similar trends were observed from the simulations, which allowed us to hypothesize that positively charged patches of the amino acids lysine and arginine in all three peptides are essential for them to interact with and translocate over the bilayer. We therefore hypothesize that electrostatic interactions are important for the interaction between the solid-supported lipid bilayers and the peptide depending on the linear charge density through the primary sequence and the positively charged patches in the sequence. The understanding of how, why, and when the cushion is formed opens up the possibility for this system to be used in the research and development of new drugs and pharmaceuticals.

This study focuses on the intrinsically disordered regulatory domain of p53 and the impact of post-translational modifications. Through fully atomistic explicit water molecular dynamics simulations, we show the wealth of information and detailed understanding that can be obtained by varying the number of phosphorylated amino acids and implementing a restriction in the conformational entropy of the N-termini of that intrinsically disordered region. The take-home message for the reader is to achieve a detailed understanding of the impact of phosphorylation with respect to (1) the conformational dynamics and flexibility, (2) structural effects, (3) protein interactivity, and (4) energy landscapes and conformational ensembles. Although our model system is the regulatory domain p53 of the tumor suppressor protein p53, this study contributes to understanding the general effects of intrinsically disordered phosphorylated proteins and the impact of phosphorylated groups, more specifically, how minor changes in the primary sequence can affect the properties mentioned above.

The pre-tetramerization loop (PTL) of the human tumor suppressor protein p53 is an intrinsically disordered region (IDR) necessary for the tetramerization process, and its flexibility contributes to the essential conformational changes needed. Although the IDR can be accurately simulated in the traditional manner of molecular dynamics (MD) with the end-to-end distance (EE dist ) unhindered, we sought to explore the effects of restraining the EE dist to the values predicted by electron microscopy (EM) and other distances. Simulating the PTL trajectory with a restrained EE dist , we found an increased agreement of nuclear magnetic resonance (NMR) chemical shifts with experiments. Additionally, we observed a plethora of secondary structures and contacts that only appear when the trajectory is restrained. Our findings expand the understanding of the tetramerization of p53 and provide insight into how mutations could make the protein impotent. In particular, our findings demonstrate the importance of restraining the EE dist in studying IDRs and how their conformations change under different conditions. Our results provide a better understanding of the PTL and the conformational dynamics of IDRs in general, which are useful for further studies regarding mutations and their effects on the activity of p53.

Integration of fiber modification step with a modern pulp mill is a resource efficient way to produce functional fibers. Motivated by the need to integrate polymer adsorption with the current pulping system, anion-specific effects in carboxymethylcellulose (CMC) adsorption have been studied. The QCM-D adsorption experiments revealed that CMC adsorption to the cellulose model surface is prone to anion-specific effects. A correlation was observed between the adsorbed CMC and the degree of hydration of the co-ions present in the magnesium salts. The presence of a chaotropic co-ion such as nitrate increased the adsorption of CMC on cellulose compared to the presence of the kosmotropic sulfate co-ion. However, anion-specificity was not significant in the case of salts containing zinc cations. The hydration of anions determines the distribution of the ions at the interface. Chaotropic ions, such as nitrates, are likely to be distributed near the chaotropic cellulose surface, causing changes in the ordering of water molecules and resulting in greater entropy gain once released from the surface, thus increasing CMC adsorption.

Coarse-graining is commonly used to decrease the computational cost of simulations. However, coarse-grained models are also considered to have lower transferability, with lower accuracy for systems outside the original scope of parametrization. Here, we benchmark a bead-necklace model and a modified Martini 2 model, both coarse-grained models, for a set of intrinsically disordered proteins, with the different models having different degrees of coarse-graining. The SOP-IDP model has earlier been used for this set of proteins; thus, those results are included in this study to compare how models with different levels of coarse-graining compare. The sometimes naive expectation of the least coarse-grained model performing best does not hold true for the experimental pool of proteins used here. Instead, it showed the least good agreement, indicating that one should not necessarily trust the otherwise intuitive notion of a more advanced model inherently being better in model choice.

Histatin 5 is a histidine-rich, intrinsically disordered, multifunctional saliva protein known to act as a first line of defense against oral candidiasis caused by Candida albicans . An earlier study showed that, upon interaction with a common model bilayer, a protein cushion spontaneously forms underneath the bilayer. Our hypothesis is that this effect is of electrostatic origin and that the observed behavior is due to proton charge fluctuations of the histidines, promoting attractive electrostatic interactions between the positively charged proteins and the anionic surfaces, with concomitant counterion release. Here we are investigating the role of the histidines in more detail by defining a library of variants of the peptide, where the former have been replaced by the pH-insensitive amino acid glutamine. By using experimental techniques such as circular dichroism, small angle X-ray scattering, quartz crystal microbalance with dissipation monitoring, and neutron reflectometry, it was determined that changing the number of histidines in the peptide sequence did not affect the structure of the peptide dissolved in solution. However, it was shown to affect the penetration depth of the peptide into the bilayer, where all variants except the one with zero histidines were found below the bilayer. A decrease in the number of histidine from the original seven to zero decreases the ability of the peptide to penetrate the bilayer, and the peptide is then also found residing within the bilayer. We hypothesize that this is due to the ability of the histidines to charge titrate, which charges up the peptide, and enables it to penetrate and translocate through the lipid bilayer.

Intrinsically disordered proteins (IDPs) have a broad energy landscape and consequently sample many different conformations in solution. The innate flexibility of IDPs is exploited in their biological function, and in many instances allows a single IDP to regulate a range of processes in vivo. Due to their highly flexible nature, characterizing the structural properties of IDPs is not straightforward. Often solution-based methods such as Nuclear Magnetic Resonance (NMR), Förster Resonance Energy Transfer (FRET), and Small-Angle X-ray Scattering (SAXS) are used. SAXS is indeed a powerful technique to study the structural and conformational properties of IDPs in solution, and from the obtained SAXS spectra, information about the average size, shape, and extent of oligomerization can be determined. In this chapter, we will introduce model-free methods that can be used to interpret SAXS data and introduce methods that can be used to interpret SAXS data beyond analytical models, for example, by using atomistic and different levels of coarse-grained models in combination with molecular dynamics (MD) and Monte Carlo simulations., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Glycolipids such as gangliosides affect the properties of lipid membranes and in extension the interactions between membranes and other biomolecules like proteins. To better understand how the properties of individual lipid molecules can contribute to shape the functional aspects of a membrane, the spatial restriction and dynamics of C-H bond segments can be measured using nuclear magnetic resonance (NMR) spectroscopy. We combine solid-state NMR spectroscopy with all-atom molecular dynamics (MD) simulations to investigate how ganglioside GM3 affects the bilayer structure and dynamics of C-H bond segments. These two methods yield reorientational correlation functions, molecular profiles of C-H bond order parameters | S CH | and effective correlation times τ e , which we compare for lipids in POPC bilayers with and without 30 mol% GM3. Our results revealed that all C-H segments of POPC reorient slower in the presence of GM3 and that the defining features of the GM3-POPC bilayer lie in the GM3 headgroup; it gives the bilayer an extended headgroup layer with high order (| S CH | up to 0.3-0.4) and slow dynamics ( τ e up to 100 ns), a character that may be mechanistically important in ganglioside interactions with other biomolecules.

This study investigates possible structural changes of an intrinsically disordered protein (IDP) when it adsorbs to a solid surface. Experiments on IDPs primarily result in ensemble averages due to their high dynamics. Therefore, molecular dynamics (MD) simulations are crucial for obtaining more detailed information on the atomistic and molecular levels. An evaluation of seven different force field and water model combinations have been applied: (A) CHARMM36IDPSFF + CHARMM-modified TIP3P, (B) CHARMM36IDPSFF + TIP4P-D, (C) CHARMM36m + CHARMM-modified TIP3P, (D) AMBER99SB-ILDN + TIP3P, (E) AMBER99SB-ILDN + TIP4P-D, (F) AMBERff03ws + TIP4P/2005, and (G) AMBER99SB-disp + disp-water. The results have been qualitatively compared with those of small-angle X-ray scattering, synchrotron radiation circular dichroism spectroscopy, and attenuated total reflectance Fourier transform infrared spectroscopy. The model IDP corresponds to the first 33 amino acids of the N-terminal of the magnesium transporter A (MgtA) and is denoted as KEIF. With a net charge of +3, KEIF is found to adsorb to the anionic synthetic clay mineral Laponite ® due to the increase in entropy from the concomitant release of counterions from the surface. The experimental results show that the peptide is largely disordered with a random coil conformation, whereas the helical content (α- and/or 3 10 -helices) increased upon adsorption. MD simulations corroborate these findings and further reveal an increase in polyproline II helices and an extension of the peptide conformation in the adsorbed state. In addition, the simulations provided atomistic resolution of the adsorbed ensemble of structures, where the arginine residues had a high propensity to form hydrogen bonds with the surface. Simulations B, E, and G showed significantly better agreement with experiments than the other simulations. Particularly noteworthy is the discovery that B and E with TIP4P-D water had superior performance to their corresponding simulations A and D with TIP3P-type water. Thus, this study shows the importance of the water model when simulating IDPs and has also provided an insight into the structural changes of surface-active IDPs induced by adsorption, which may play an important role in their function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Koder Hamid, Månsson, Meklesh, Persson and Skepö.)

Our aim was to understand mechanisms for clustering and cross-linking of gliadins, a wheat seed storage protein type, monomeric in native state, but incorporated in network while processed. The mechanisms were studied utilizing spectroscopy and high-performance liquid chromatography on a gliadin-rich fraction, in vitro produced α-gliadins, and synthetic gliadin peptides, and by coarse-grained modelling, Monte Carlo simulations and prediction algorithms. In solution, gliadins with α-helix structures (dip at 205 nm in CD) were primarily present as monomeric molecules and clusters of gliadins (peaks at 650- and 700-s on SE-HPLC). At drying, large polymers (R g 90.3 nm by DLS) were formed and β-sheets increased (14% by FTIR). Trained algorithms predicted aggregation areas at amino acids 115-140, 150-179, and 250-268, and induction of liquid-liquid phase separation at P- and Poly-Q-sequences (Score = 1). Simulations showed that gliadins formed polymers by tail-to-tail or a hydrophobic core (Kratky plots and R ee  = 35 and 60 for C- and N-terminal). Thus, the N-terminal formed clusters while the C-terminal formed aggregates by disulphide and lanthionine bonds, with favoured hydrophobic clustering of similar/exact peptide sections (synthetic peptide mixtures on SE-HPLC). Mechanisms of clustering and cross-linking of the gliadins presented here, contribute ability to tailor processing results, using these proteins., (Copyright © 2022. Published by Elsevier B.V.)

Intrinsically disordered proteins (IDPs) are proteins that, in comparison with globular/structured proteins, lack a distinct tertiary structure. Here, we use the model IDP, Histatin 5, for studying its dynamical properties under self-crowding conditions with quasi-elastic neutron scattering in combination with full atomistic molecular dynamics (MD) simulations. The aim is to determine the effects of crowding on the center-of-mass diffusion as well as the internal diffusive behavior. The diffusion was found to decrease significantly, which we hypothesize can be attributed to some degree of aggregation at higher protein concentrations, (≥100 mg/mL), as indicated by recent small-angle X-ray scattering studies. Temperature effects are also considered and found to, largely, follow Stokes-Einstein behavior. Simple geometric considerations fail to accurately predict the rates of diffusion, while simulations show semiquantitative agreement with experiments, dependent on assumptions of the ratio between translational and rotational diffusion. A scaling law that previously was found to successfully describe the behavior of globular proteins was found to be inadequate for the IDP, Histatin 5. Analysis of the MD simulations show that the width of the distribution with respect to diffusion is not a simplistic mirroring of the distribution of radius of gyration, hence, displaying the particular features of IDPs that need to be accounted for.

It is well-known that an increasing proportion of proteins, protein regions, and partners of globular proteins are being recognized as having an intrinsic disorder, and therefore, not adopting a single three-dimensional structure in solution. For these proteins, small-angle X-ray scattering (SAXS) has become a premier method for examination, since it can provide information about the ensemble of the structural conformations as well as the intermolecular interactions. SAXS measurements can be performed from low to high protein concentrations under different physicochemical properties of the solution. The focus of this chapter is to introduce the basics of how to use SAXS for protein samples, for new and less experienced users, in a simple and concise manner, with emphasis on highly flexible proteins and regions. Methodological aspects in the sample preparation, experiment design, and data collection stages are raised that should be considered prior to attempting SAXS experiments. This is to ensure that high-quality SAXS data is obtained that enables accurate analysis. However, many of the points raised will also be worth considering for SAXS experiments of globular proteins., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Intrinsically disordered proteins are involved in many biological processes such as signaling, regulation, and recognition. A common strategy to regulate their function is through phosphorylation, as it can induce changes in conformation, dynamics, and interactions with binding partners. Although phosphorylated intrinsically disordered proteins have received increased attention in recent years, a full understanding of the conformational and structural implications of phosphorylation has not yet been achieved. Here, we present all-atom molecular dynamics simulations of five disordered peptides originated from tau, statherin, and β-casein, in both phosphorylated and non-phosphorylated state, to compare changes in global dimensions and structural elements, in an attempt to gain more insight into the controlling factors. The changes are in qualitative agreement with experimental data, and we observe that the net charge is not enough to predict the impact of phosphorylation on the global dimensions. Instead, the distribution of phosphorylated and positively charged residues throughout the sequence has great impact due to the formation of salt bridges. In statherin, a preference for arginine-phosphoserine interaction over arginine-tyrosine accounts for a global expansion, despite a local contraction of the phosphorylated region, which implies that also non-charged residues can influence the effect of phosphorylation.

Five peptides previously suggested to possess polyproline II (PPII) structure have here been investigated by using atomistic molecular dynamics simulations to compare how well four different force fields known for simulating intrinsically disordered proteins relatively well (Amber ff99SB-disp, Amber ff99SB-ILDN, CHARM36IDPSFF, and CHARMM36m) can capture this secondary structure element. The results revealed that all force fields sample PPII structures but to different extents and with different propensities toward other secondary structure elements, in particular, the β-sheet and "random coils". A cluster analysis of the simulations of histatin 5 also revealed that the conformational ensembles of the force fields are quite different. We compared the simulations to circular dichroism and nuclear magnetic resonance spectroscopy experiments and conclude that further experiments and methods for interpreting them are needed to assess the accuracy of force fields in determining PPII structure.

Phosphorylation is a common post-translational modification among intrinsically disordered proteins and regions, which helps regulate function by changing the protein conformations, dynamics, and interactions with binding partners. To fully comprehend the effects of phosphorylation, computer simulations are a helpful tool, although they are dependent on the accuracy of the force field used. Here, we compared the conformational ensembles produced by Amber ff99SB-ILDN+TIP4P-D and CHARMM36m, for four phosphorylated disordered peptides ranging in length from 14-43 residues. CHARMM36m consistently produced more compact conformations with a higher content of bends, mainly due to more stable salt bridges. Based on comparisons with experimental size estimates for the shortest and longest peptide, CHARMM36m appeared to overestimate the compactness. The difference between the force fields was largest for the peptide showing the greatest separation between positively charged and phosphorylated residues, in line with the importance of charge distribution. For this peptide, the conformational ensemble did not change significantly upon increasing the ionic strength from 0 mM to 150 mM, despite a reduction of the salt-bridging probability in the CHARMM36m simulations, implying that salt concentration has negligible effects in this study.

In this work, we have synthesized polystyrene particles that carry short end-grafted polyethylene glycol (PEG) chains. We then added dissolved 100 kDa PEG polymers and monitored potential flocculation by confocal microscopy. Qualitative predictions, based on previous theoretical developments in this field (Xie, F.; et al. Soft Matter 2016 , 12 , 658), suggest a non-monotonic temperature response. These theories propose that the "free" (dissolved) polymers will mediate attractive depletion interactions at room temperature, with a concomitant clustering/flocculation at a sufficiently high polymer concentration. At high temperatures, where the solvent is poorer, this is predicted to be replaced by attractive bridging interactions, again resulting in particle condensation. Interestingly enough, our theoretical framework, based on classical density functional theory, predicts an intermediate temperature regime where the polymer-mediated interactions are repulsive ! This obviously implies a homogeneous dispersion in this regime. These qualitative predictions have been experimentally tested and confirmed in this work, where flocs of particles start to form at room temperature for a high enough polymer dosage. At temperatures near 45 °C, the flocs redisperse, and we obtain a homogeneous sample. However, samples at about 75 °C will again display clusters and eventually phase separation. Using results from these studies, we have been able to fine-tune parameters of our coarse-grained theoretical model, resulting in predictions of temperature-dependent stability that display semiquantitative accuracy. A crucial aspect is that under "intermediate" conditions, where the polymers neither adsorb nor desorb at the particle surfaces, the polymer-mediated equilibrium interaction is repulsive ., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Re-entrant condensation results in the formation of a condensed protein regime between two critical ion concentrations. The process is driven by neutralization and inversion of the protein charge by oppositely charged ions. Re-entrant condensation of cationic proteins by the polyvalent anions, pyrophosphate and tripolyphosphate, has previously been observed, but not for citrate, which has similar charge and size compared to the polyphosphates. Therefore, besides electrostatic interactions, other specific interactions between the polyphosphate ions and proteins must contribute. Here, we show that additional attractive interactions between arginine and tripolyphosphate determine the re-entrant condensation and decondensation boundaries of the cationic, intrinsically disordered saliva protein, histatin 5. Furthermore, we show by small-angle X-ray scattering (SAXS) that polyvalent anions cause compaction of histatin 5, as would be expected based solely on electrostatic interactions. Hence, we conclude that arginine-phosphate-specific interactions not only regulate solution properties but also influence the conformational ensemble of histatin 5, which is shown to vary with the number of arginine residues. Together, the results presented here provide further insight into an organizational mechanism that can be used to tune protein interactions in solution of both naturally occurring and synthetic proteins.

The Protein Ensemble Database (PED) (https://proteinensemble.org), which holds structural ensembles of intrinsically disordered proteins (IDPs), has been significantly updated and upgraded since its last release in 2016. The new version, PED 4.0, has been completely redesigned and reimplemented with cutting-edge technology and now holds about six times more data (162 versus 24 entries and 242 versus 60 structural ensembles) and a broader representation of state of the art ensemble generation methods than the previous version. The database has a completely renewed graphical interface with an interactive feature viewer for region-based annotations, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. High quality of the data is guaranteed by a new submission process, which combines both automatic and manual evaluation steps. A team of biocurators integrate structured metadata describing the ensemble generation methodology, experimental constraints and conditions. A new search engine allows the user to build advanced queries and search all entry fields including cross-references to IDP-related resources such as DisProt, MobiDB, BMRB and SASBDB. We expect that the renewed PED will be useful for researchers interested in the atomic-level understanding of IDP function, and promote the rational, structure-based design of IDP-targeting drugs., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Hypothesis: Colloidal particles that interact via a long-ranged repulsive barrier in combination with a very short-ranged attractive minimum can "polymerize" to form highly anisotropic structures. Motivated by previous experimental achievements in non-aqueous solvents, and recent theoretical predictions, we hypothesize that it is possible to construct clusters that resemble linear or branched polymers, in aqueous solution. If these clusters are not too large, they may even remain dispersed, but even if they grow large enough to sediment, they may be collected and used in future applications., Experiments: In this work, we specifically synthesize poly (ethylene glycol) (PEG) chains, grafted onto poly (styrene) (PS) particles in aqueous solution, and adjust the conditions so that strongly anisotropic and isolated polymer-like clusters are formed. These conditions include a very low ionic strength (the particles are weakly charged), a relatively high temperature, and a low particle concentration. An important criterion is that the particle size is large enough to admit structural analyses via confocal laser scanning microscopy (CLSM). We have furthermore utilized Metropolis Monte Carlo (MC) simulation to generate theoretical predictions of these cluster formations. We have conducted such simulations of 3D as well as 2D systems, where the latter is also relevant, given that the clusters sometimes deposit onto the glass surfaces upon imaging. A simplistic particle-particle potential of mean force is adopted for the simulations, but we also invoke a more elaborate theoretical model, to demonstrate that similar interactions can be obtained when the grafted chains are treated explicitly., Findings: According to our Zeta potential measurements, the particles indeed carry a weak negative charge, presumably due to ion specific adsorption. Furthermore, by ensuring that the ionic strength is very low, with a Debye length similar to the particle size, we could use temperature to control the hydrophobicity of the grafted PEG layer, and thus the strength of the short-ranged attraction. We were indeed able to establish highly anisotropic structures, that resemble linear or branched polymers, which we could image by CLSM. The average degree of polymerization could be adjusted by a variation of the particle concentration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intrinsically disordered proteins (IDP) are proteins that sample a heterogeneous ensemble of conformers in solution. An estimated 25-30% of all eukaryotic proteins belong to this class. In vivo , IDPs function under conditions that are highly crowded by other biological macromolecules. Previous research has highlighted that the presence of crowding agents can influence the conformational ensemble sampled by IDPs, resulting in either compaction or expansion. The effects of self-crowding of the disordered protein Histatin 5 has, in an earlier study, been found to have limited influence on the conformational ensemble. In this study, it is examined whether the short chain length of Histatin 5 can explain the limited effects of crowding observed, by introducing (Histatin 5) 2 , a tandem repeat of Histatin 5. By utilizing small-angle X-ray scattering, it is shown that the conformational ensemble is conserved at high protein concentrations, in resemblance with Histatin 5, although with a lowered protein concentration at which aggregation arises. Under dilute conditions, atomistic molecular dynamics and coarse-grained Monte Carlo simulations, as well as an established scaling law, predicted more extended conformations than indicated by experimental data, hence implying that (Histatin 5) 2 does not behave as a self-avoiding random walk.

Enterohemorrhagic and enteropathogenic Escherichia coli are among the most important food-borne pathogens, posing a global health threat. The virulence factor intimin is essential for the attachment of pathogenic E. coli to the intestinal host cell. Intimin consists of four extracellular bacterial immunoglobulin-like (Big) domains, D00-D2, extending into the fifth lectin subdomain (D3) that binds to the Tir-receptor on the host cell. Here, we present the crystal structures of the elusive D00-D0 domains at 1.5 Å and D0-D1 at 1.8 Å resolution, which confirms that the passenger of intimin has five distinct domains. We describe that D00-D0 exhibits a higher degree of rigidity and D00 likely functions as a juncture domain at the outer membrane-extracellular medium interface. We conclude that D00 is a unique Big domain with a specific topology likely found in a broad range of other inverse autotransporters. The accumulated data allows us to model the complete passenger of intimin and propose functionality to the Big domains, D00-D0-D1, extending directly from the membrane.

Hypothesis: The stability of colloidal dispersions in the presence of multivalent ions depends strongly on the electrostatic interactions between the suspended particles. Of particular interest are colloidal particles having dimensions in the nanometric range and with an anisotropic shape due to its high surface area per unit mass, for example clay, which has the key characteristic of a negatively charged surface, surrounded by an oppositely charged rim., Experiments: In this study, we investigate the interactions in nanoplatelet dispersions for the model system of Laponite® clay with addition of mono- and multivalent salt. Molecular dynamics simulations with enhanced umbrella sampling have been utilised in combination with the experimental techniques of zeta-potential measurements, dynamic light scattering, and transmission electron microscopy., Findings: It was observed that tactoid formation and tactoidal dissolution due to overcharging occur upon the addition of trivalent salt. The overcharging effect was captured from calculated potential of mean force and confirmed from the zeta-potential, which changed sign from negative to positive when increasing the stoichiometric charge-ratio between the positive salt ions and the clay. Consequently, the gained information could provide useful physical insight of nanoplatelet interactions in the presence of multivalent ions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gluten protein crosslinking is a predetermined process where specific intra- and intermolecular disulfide bonds differ depending on the protein and cysteine motif. In this article, all-atom Monte Carlo simulations were used to understand the formation of disulfide bonds in gliadins and low molecular weight glutenin subunits (LMW-GS). The two intrinsically disordered proteins appeared to contain mostly turns and loops and showed "self-avoiding walk" behavior in water. Cysteine residues involved in intramolecular disulfide bonds were located next to hydrophobic peptide sections in the primary sequence. Hydrophobicity of neighboring peptide sections, synthesis chronology, and amino acid chain flexibility were identified as important factors in securing the specificity of intramolecular disulfide bonds formed directly after synthesis. The two LMW-GS cysteine residues that form intermolecular disulfide bonds were positioned next to peptide sections of lower hydrophobicity, and these cysteine residues are more exposed to the cytosolic conditions, which influence the crosslinking behavior. In addition, coarse-grained Monte Carlo simulations revealed that the protein folding is independent of ionic strength. The potential molecular behavior associated with disulfide bonds, as reported here, increases the biological understanding of seed storage protein function and provides opportunities to tailor their functional properties for different applications.

In this article, it is shown that by exposing commonly used lipids for biomembrane mimicking studies, to a solution containing the histidine-rich intrinsically disordered protein histatin 5, a protein cushion spontaneously forms underneath the bilayer. The underlying mechanism is attributed to have an electrostatic origin, and it is hypothesized that the observed behavior is due to proton charge fluctuations promoting attractive electrostatic interactions between the positively charged proteins and the anionic surfaces, with concomitant counterion release. Hence, we anticipate that this novel "green" approach of forming cushioned bilayers can be an important tool to mimic the cell membrane without the disturbance of the solid substrate, thereby achieving a further understanding of protein-cell interactions.

Magnesium transporter A (MgtA) is an active transporter responsible for importing magnesium ions into the cytoplasm of prokaryotic cells. This study focuses on the peptide corresponding to the intrinsically disordered N-terminal region of MgtA, referred to as KEIF. Primary-structure and bioinformatic analyses were performed, followed by studies of the undisturbed single chain using a combination of techniques including small-angle X-ray scattering, circular dichroism spectroscopy, and atomistic molecular-dynamics simulations. Moreover, interactions with large unilamellar vesicles were investigated by using dynamic light scattering, laser Doppler velocimetry, cryogenic transmission electron microscopy, and circular dichroism spectroscopy. KEIF was confirmed to be intrinsically disordered in aqueous solution, although extended and containing little β -structure and possibly PPII structure. An increase of helical content was observed in organic solvent, and a similar effect was also seen in aqueous solution containing anionic vesicles. Interactions of cationic KEIF with anionic vesicles led to the hypothesis that KEIF adsorbs to the vesicle surface through electrostatic and entropic driving forces. Considering this, there is a possibility that the biological role of KEIF is to anchor MgtA in the cell membrane, although further investigation is needed to confirm this hypothesis.

We present a scheme for transferring conformational degrees of freedom from all-atom (AA) simulations of an intrinsically disordered protein (IDP) to coarse-grained (CG) Monte Carlo (MC) simulations using conformational swap moves. AA simulations of a single histatin 5 peptide in water were used to obtain a structural ensemble, which is reweighted in a CGMC simulation in the presence of a negatively charged surface. For efficient sampling, the AA trajectory was condensed using two approaches: RMSD clustering (based on the root-mean-square difference in atom positions) and a "naı̈ve" truncation, where only every 100th frame of the trajectory was included in the library. The results show that even libraries with few structures well reproduce the radius of gyration and interaction free energy as functions of the distance from the surface. We further observe that the surface slightly promotes the secondary structure of histatin 5 and more so if using explicit surface charges rather than smeared charges.

Phosphorylation is one of the most abundant types of post-translational modifications of intrinsically disordered proteins (IDPs). This study examines the conformational changes in the 15-residue-long N-terminal fragment of the IDP statherin upon phosphorylation, using computer simulations with two different force fields: AMBER ff99SB-ILDN and CHARMM36m. The results from the simulations are compared with experimental small-angle X-ray scattering (SAXS) and circular dichroism data. In the unphosphorylated state, the two force fields are in excellent agreement regarding global structural properties such as size and shape. However, they exhibit some differences in the extent and type of the secondary structure. In the phosphorylated state, neither of the force fields performs well compared to the experimental data. Both force fields show a compaction of the peptide upon phosphorylation, greater than what is seen in SAXS experiments, although they differ in the local structure. While the CHARMM force field increases the fraction of bends in the peptide as a response to strong interactions between the phosphorylated residues and arginines, the AMBER force field shows an increase of the helical content in the N-terminal part of the peptide, where the phosphorylated residues reside, in better agreement with circular dichroism results.

When a biomaterial is inserted into the body, proteins rapidly adsorb onto its surface, creating a conditioning protein film that functions as a link between the implant and adhering cells. Depending on the nano-roughness of the surface, proteins will adsorb in different amounts, with different conformations and orientations, possibly affecting the subsequent attachment of cells to the surface. Thus, modifications of the surface nanotopography of an implant may prevent biomaterial-associated infections. Fibrinogen is of particular importance since it contains adhesion epitopes that are recognized by both eukaryotic and prokaryotic cells, and can therefore influence the adhesion of bacteria. The aim of this study was to model adsorption of fibrinogen to smooth or nanostructured silica surfaces in an attempt to further understand how surface nanotopography may affect the orientation of the adsorbed fibrinogen molecule. We used a coarse-grained model, where the main body of fibrinogen (visible in the crystal structure) was modeled as rigid and the flexible α C-chains (not visible in the crystal structure) were modeled as completely disordered. We found that the elongated fibrinogen molecule preferably adsorbs in such a way that it protrudes further into solution on a nanostructured surface compared to a flat one. This implicates that the orientation on the flat surface increases its bio-availability.

There is a great interest within the research community to understand the structure-function relationship for intrinsically disordered proteins (IDPs); however, the heterogeneous distribution of conformations that IDPs can adopt limits the applicability of conventional structural biology methods. Here, scattering techniques, such as small-angle X-ray scattering, can contribute. In this chapter, we will describe how to make a model-free determination of the radius of gyration by using two different approaches, the Guinier analysis and the pair distance distribution function. The ATSAS package (Franke et al., J Appl Crystallogr 50:1212-1225, 2017) has been used for the evaluation, and throughout the chapter, different examples will be given to illustrate the discussed phenomena, as well as the pros and cons of using the different approaches.

Intrinsically disordered proteins (IDPs) adopt heterogeneous conformational ensembles in solution. The properties of the conformational ensemble are dependent upon the solution conditions, including the presence of ions, temperature, and crowding, and often directly impact biological function. Many in vitro investigations focus on the properties of IDPs under dilute conditions, rather than the crowded environment found in vivo . Due to their heterogeneous nature, the study of IDPs under crowded conditions is challenging both experimentally and computationally. Despite this, such studies are worth pursuing due to the insight gained into biologically relevant phenomena. Here, we study the highly charged IDP Histatin 5 under self-crowded conditions in low and high salt conditions. A combination of small-angle X-ray scattering and different simulation models, spanning a range of computational complexity and detail, is used. Most models are found to have limited application when compared to results from experiments. The best performing model is the highly coarse-grained, bead-necklace model. This model shows that Histatin 5 has a conserved radius of gyration and a decreasing flexibility with increasing protein concentration. Due to its computational efficiency, we propose that it is a suitable model to study crowded IDP solutions, despite its simplicity.

The addition of polyelectrolytes (PEs) to suspensions of charged colloids, such as nanoplatelets (NPs), is of great interest due to their specific feature of being either a stabilizing or a destabilizing agent. Here, the complexation between a PE and oppositely charged NPs is studied utilizing coarse-grained molecular dynamics simulations based on the continuum model. The complex formation is evaluated with respect to the stoichiometric charge-ratio within the system, as well as by the alternation of the chain properties. It is found that the formed complexes can possess either an extended or a compact shape. Moreover, it is observed that the chain can become overcharged by the oppositely charged NPs. With an increase in chain length, or a decrease in chain flexibility, the complex obtains a more extended shape, where the NPs are less tightly bound to the PE. The latter is also true when reducing the total charge of the chain by varying the linear charge density, whereas in this case, the chain contracts. With our coarse-grained model and molecular dynamics simulations, we are able to predict the composition and the shape of the formed complex and how it is affected by the characteristics of the chain. The take-home message is that the complexation between PEs and NPs results in a versatile and rich state diagram, which indeed is difficult to predict, and dependent on the properties of the chain and the model used. Thus, we propose that the present model can be a useful tool to achieve an understanding of the PE-NPs complexation, a system commonly used in industrial and in technological processes.

Clays can be synthesised to have specific functional properties, which have been exploited in a range of industrial processes. A key characteristic of clay is the presence of a negatively charged surface, surrounded by an oppositely charged rim. Because of that, clays are able to sequester cationic compounds resulting in the formation of ordered layered structures, known as tactoids. Recent research has highlighted the possibility of utilising clay as a drug delivery compound for cationic peptides. Here, we investigate the process of intercalation by using the highly cationic peptide deca-arginine, and the synthetic clay Laponite, in aqueous suspensions with 2.5 wt% Laponite, and varying peptide concentrations. Small-angle X-ray scattering experiments show that tactoids are formed as a function of deca-arginine concentration in the dispersion, and for an excess of peptide, i.e. above a matched charge-ratio between the peptide and clay, the growth of the tactoids is limited, resulting in tactoidal dissolution. Zeta-potential measurements confirm that the observed dissolution is caused by overcharging of the platelets. By employing coarse-grained molecular dynamics simulations based on the continuum model, we are able to predict the tactoid formation, the growth, and the dissolution, in agreement with experimental results. We propose that the present simulation method can be a useful tool to tune peptide and clay characteristics to optimise and determine the extent of intercalation by cationic peptides of therapeutic interest., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intrinsically disordered proteins (IDPs) can form functional oligomers and in some cases, insoluble disease related aggregates. It is therefore vital to understand processes and mechanisms that control pathway distribution. Divalent cations including Zn 2+ can initiate IDP oligomerisation through the interaction with histidine residues but the mechanisms of doing so are far from understood. Here we apply a multi-disciplinary approach using small angle X-ray scattering, nuclear magnetic resonance spectroscopy, calorimetry and computations to show that that saliva protein Histatin 5 forms highly dynamic oligomers in the presence of Zn 2+ . The process is critically dependent upon interaction between Zn 2+ ions and distinct histidine rich binding motifs which allows for thermodynamic switching between states. We propose a molecular mechanism of oligomerisation, which may be generally applicable to other histidine rich IDPs. Finally, as Histatin 5 is an important saliva component, we suggest that Zn 2+ induced oligomerisation may be crucial for maintaining saliva homeostasis.

Attractive interactions between intrinsically disordered proteins can be crucial for the functionality or, on the contrary, lead to the formation of harmful aggregates. For obtaining a molecular understanding of intrinsically disordered proteins and their interactions, computer simulations have proven to be a valuable complement to experiments. In this study, we present a coarse-grained model and its applications to a system dominated by attractive interactions, namely, the self-association of the saliva protein Statherin. SAXS experiments show that Statherin self-associates with increased protein concentration, and that both an increased temperature and a lower ionic strength decrease the size of the formed complexes. The model captures the observed trends and provides insight into the size distribution. Hydrophobic interaction is considered to be the major driving force of the self-association, while electrostatic repulsion represses the growth. In addition, the model suggests that the decrease of association number with increased temperature is of entropic origin., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Solution techniques such as small-angle X-ray scattering (SAXS) play a central role in structural studies of intrinsically disordered proteins (IDPs); yet, due to low resolution, it is generally necessary to combine SAXS with additional experimental sources of data and to use molecular simulations. Computational methods for the calculation of theoretical SAXS intensity profiles can be separated into two groups, depending on whether the solvent is modeled implicitly as continuous electron density or considered explicitly. The former offers reduced computational cost but requires the definition of a number of free parameters to account for, for example, the excess density of the solvation layer. Overfitting can thus be an issue, particularly when the structural ensemble is unknown. Here, we investigate and show how small variations of the contrast of the hydration shell, δρ, severely affect the outcome, analysis and interpretation of computed SAXS profiles for folded and disordered proteins. For both the folded and disordered proteins studied here, using a default δρ may, in some cases, result in the calculation of non-representative SAXS profiles, leading to an overestimation of their size and a misinterpretation of their structural nature. The solvation layer of the different IDP simulations also impacts their size estimates differently, depending on the protein force field used. The same is not true for the folded protein simulations, suggesting differences in the solvation of the two classes of proteins, and indicating that different force fields optimized for IDPs may cause expansion of the polypeptide chain through different physical mechanisms., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In this study, we have used the coarse-grained model developed for the intrinsically disordered saliva protein (IDP) Histatin 5, on an experimental selection of monomeric IDPs, and we show that the model is generally applicable when electrostatic interactions dominate the intra-molecular interactions. Experimental and theoretically calculated small-angle X-ray scattering data are presented in the form of Kratky plots, and discussions are made with respect to polymer theory and the self-avoiding walk model. Furthermore, the impact of electrostatic interactions is shown and related to estimations of the conformational ensembles obtained from computer simulations and "Flexible-meccano." Special attention is given to the form factor and how it is affected by the salt concentration, as well as the approximation of using the form factor obtained under physiological conditions to obtain the structure factor., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Films engineered to control the transport of liquids are widely used through society. Examples include barriers in packaging, wound care products, and controlled release coatings in pharmaceutics. When observed at the macroscopic scale such films commonly appear homogeneous, however, a closer look reveals a complex nano- and microstructure that together with the chemical properties of the different domains control the transport properties. In this review we compare and discuss macroscopic transport properties, measured using the straightforward, yet highly powerful technique "modified Ussing chambers", also denoted side-by-side diffusion cells, for a wide range of structured polymer films and composites. We also discuss and compare the macroscopic observations and conclusions on materials properties with that of lattice Boltzmann simulations of transport properties based on underlying material structure and chemistry. The survey of the field: (i) highlights the use and power of modified Ussing Chambers for determining liquid transport properties of polymer films, (ii) demonstrates the predictability in both directions between macroscopic observations of transport using modified Ussing chambers and structure-based simulations, and (iii) provides experimental and theoretical insights regarding the transport-determining properties of structured polymer films and composites., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The structural properties, and the intracrystalline swelling of Na + -, and Ca 2+ -montmorillonite (Na-, and Ca-mmt) have been investigated as an effect of decreasing the relative permittivity of the solvent, i.e. from water to ethanol (EtOH), utilizing the experimental techniques; small angle X-ray scattering (SAXS) and osmotic pressure measurements. The experimental data were compared with the continuum model, utilizing coarse-grained molecular dynamics bulk simulations, Monte Carlo simulations of two infinite parallel surfaces corresponding to two clay platelets, and the strong coupling theory. It was found that it is possible to tune the electrostatic interactions to obtain a transition from a repulsive to an attractive system for the Na-mmt by increasing the EtOH concentration, i.e. the Bjerrum length increases, and hence, the attractive ion-ion correlation forces are enhanced. A qualitative agreement was observed between the simulations and the experimental results. Moreover, a non-monotonic behavior of the intracrystalline swelling of Ca-mmt as a function of EtOH concentration was captured experimentally, where an increase in the osmotic pressure, and hence, an increase in the d-spacing was found at low concentrations, indicating that repulsive short-ranged interactions dominate in the system. Theoretically, the non-monotonic behavior could not be captured with the continuum model, probably due to the limitation that the electrostatic interactions solely enters the Hamiltonian via the Bjerrum length., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Small-angle X-ray scattering (SAXS) measurements reveal a striking difference in intermolecular interactions between two short highly charged peptides-deca-arginine (R10) and deca-lysine (K10). Comparison of SAXS curves at high and low salt concentration shows that R10 self-associates, while interactions between K10 chains are purely repulsive. The self-association of R10 is stronger at lower ionic strengths, indicating that the attraction between R10 molecules has an important electrostatic component. SAXS data are complemented by NMR measurements and potentials of mean force between the peptides, calculated by means of umbrella-sampling molecular dynamics (MD) simulations. All-atom MD simulations elucidate the origin of the R10-R10 attraction by providing structural information on the dimeric state. The last two C-terminal residues of R10 constitute an adhesive patch formed by stacking of the side chains of two arginine residues and by salt bridges formed between the like-charge ion pair and the C-terminal carboxyl groups. A statistical analysis of the Protein Data Bank reveals that this mode of interaction is a common feature in proteins., Competing Interests: The authors declare no conflict of interest.