Testing mixing rules for structural and dynamical quantities in multi-component crowded protein solutions.

Crowding effects significantly influence the phase behavior and the structural and dynamic properties of the concentrated protein mixtures present in the cytoplasm of cells or in the blood serum. This poses enormous difficulties for our theoretical understanding and our ability to predict the behavior of these systems. While the use of course grained colloid-inspired models allows us to reproduce the key physical solution properties of concentrated monodisperse solutions of individual proteins, we lack corresponding theories for complex polydisperse mixtures. Here, we test the applicability of simple mixing rules in order to predict solution properties of protein mixtures. We use binary mixtures of the well-characterized bovine eye lens proteins α and γB crystallin as model systems. Combining microrheology with static and dynamic scattering techniques and observations of the phase diagram for liquid-liquid phase separation, we show that reasonably accurate descriptions are possible for macroscopic and mesoscopic signatures, while information on the length scale of the individual protein size requires more information on cross-component interaction.

Multiscale Structural Insight into Dairy Products and Plant-Based Alternatives by Scattering and Imaging Techniques.

Dairy products and plant-based alternatives have a large range of structural features from atomic to macroscopic length scales. Scattering techniques with neutrons and X-rays provide a unique view into this fascinating world of interfaces and networks provided by, e.g., proteins and lipids. Combining these scattering techniques with a microscopic view into the emulsion and gel systems with environmental scanning electron microscopy (ESEM) assists in a thorough understanding of such systems. Different dairy products, such as milk, or plant-based alternatives, such as milk-imitating drinks, and their derived or even fermented products, including cheese and yogurt, are characterized in terms of their structure on nanometer- to micrometer-length scales. For dairy products, the identified structural features are milk fat globules, casein micelles, CCP nanoclusters, and milk fat crystals. With increasing dry matter content in dairy products, milk fat crystals are identified, whereas casein micelles are non-detectable due to the protein gel network in all types of cheese. For the more inhomogeneous plant-based alternatives, fat crystals, starch structures, and potentially protein structures are identified. These results may function as a base for improving the understanding of dairy products and plant-based alternatives, and may lead to enhanced plant-based alternatives in terms of structure and, thus, sensory aspects such as mouthfeel and texture.

Determination of Cooperativity Length in a Glass-Forming Polymer.

To describe the properties of glass-forming liquids, the concepts of a cooperativity length or the size of cooperatively rearranging regions are widely employed. Their knowledge is of outstanding importance for the understanding of both thermodynamic and kinetic properties of the systems under consideration and the mechanisms of crystallization processes. By this reason, methods of experimental determination of this quantity are of outstanding importance. Proceeding in this direction, we determine the so-called cooperativity number and, based on it, the cooperativity length by experimental measurements utilizing AC calorimetry and quasi-elastic neutron scattering (QENS) at comparable times. The results obtained are different in dependence on whether temperature fluctuations in the considered nanoscale subsystems are either accounted for or neglected in the theoretical treatment. It is still an open question, which of these mutually exclusive approaches is the correct one. As shown in the present paper on the example of poly(ethyl methacrylate) (PEMA), the cooperative length of about 1 nm at 400 K and a characteristic time of ca. 2 µs determined from QENS coincide most consistently with the cooperativity length determined from AC calorimetry measurements if the effect of temperature fluctuations is incorporated in the description. This conclusion indicates that-accounting for temperature fluctuations-the characteristic length can be derived by thermodynamic considerations from the specific parameters of the liquid at the glass transition and that temperature does fluctuate in small subsystems.

MembraneDyn: simulating the dynamics of supported membrane stacks on the nanosecond timescale.

The static structure factor and the undulation dynamics of a solid-supported membrane stack have previously been calculated by Romanov and Ul'yanov [Romanov & Ul'yanov (2002). Phys. Rev. E, 66, 061701]. Based on this prior work, the calculation has been extended to cover the membrane dynamics, i.e. the intermediate scattering function as a Fourier transform of the van Hove correlation function of the membrane stack. Fortran code which calculates the intermediate scattering function for a membrane stack on a solid support is presented. It allows the static and dynamic scattering functions to be calculated according to the derivation of Romanov and Ul'yanov. The physical properties of supported phospholipid bilayers can be examined in this way and the results can be directly compared with results obtained from grazing-incidence neutron spin-echo spectroscopy experiments.

Variation of Structural and Dynamical Flexibility of Myelin Basic Protein in Response to Guanidinium Chloride.

Myelin basic protein (MBP) is intrinsically disordered in solution and is considered as a conformationally flexible biomacromolecule. Here, we present a study on perturbation of MBP structure and dynamics by the denaturant guanidinium chloride (GndCl) using small-angle scattering and neutron spin-echo spectroscopy (NSE). A concentration of 0.2 M GndCl causes charge screening in MBP resulting in a compact, but still disordered protein conformation, while GndCl concentrations above 1 M lead to structural expansion and swelling of MBP. NSE data of MBP were analyzed using the Zimm model with internal friction (ZIF) and normal mode (NM) analysis. A significant contribution of internal friction was found in compact states of MBP that approaches a non-vanishing internal friction relaxation time of approximately 40 ns at high GndCl concentrations. NM analysis demonstrates that the relaxation rates of internal modes of MBP remain unaffected by GndCl, while structural expansion due to GndCl results in increased amplitudes of internal motions. Within the model of the Brownian oscillator our observations can be rationalized by a loss of friction within the protein due to structural expansion. Our study highlights the intimate coupling of structural and dynamical plasticity of MBP, and its fundamental difference to the behavior of ideal polymers in solution.

Nanosecond structural dynamics of intrinsically disordered ß-casein micelles by neutron spectroscopy.

ß-casein undergoes a reversible endothermic self-association, forming protein micelles of limited size. In its functional state, a single ß-casein monomer is unfolded, which creates a high structural flexibility, which is supposed to play a major role in preventing the precipitation of calcium phosphate particles. We characterize the structural flexibility in terms of nanosecond molecular motions, depending on the temperature by quasielastic neutron scattering. Our major questions are: Does the self-association reduce the chain flexibility? How does the dynamic spectrum of disordered caseins differ from a compactly globular protein? How does the dynamic spectrum of ß-casein in solution differ from that of a protein in hydrated powder states? We report on two relaxation processes on a nanosecond and a sub-nanosecond timescale for ß-casein in solution. Both processes are analyzed by Brownian oscillator model, by which the spring constant can be defined in the isotropic parabolic potential. The slower process, which is analyzed by neutron spin echo, seems a characteristic feature of the unfolded structure. It requires bulk solvent and is not seen in hydrated protein powders. The faster process, which is analyzed by neutron backscattering, has a smaller amplitude and requires hydration water, which is also observed with folded proteins in the hydrated state. The self-association had no significant influence on internal relaxation, and thus, a ß-casein protein monomer flexibility is preserved in the micelle. We derive spring constants of the faster and slower motions of ß-caseins in solution and compared them with those of some proteins in various states (folded or hydrated powder).

Inelastic neutron scattering analysis with time-dependent Gaussian-field models.

Converting neutron scattering data to real-space time-dependent structures can only be achieved through suitable models, which is particularly challenging for geometrically disordered structures. We address this problem by introducing time-dependent clipped Gaussian field models. General expressions are derived for all space- and time-correlation functions relevant to coherent inelastic neutron scattering for multiphase systems and arbitrary scattering contrasts. Various dynamic models are introduced that enable one to add time-dependence to any given spatial statistics, as captured, e.g., by small-angle scattering. In a first approach, the Gaussian field is decomposed into localized waves that are allowed to fluctuate in time or to move either ballistically or diffusively. In a second approach, a dispersion relation is used to make the spectral components of the field time-dependent. The various models lead to qualitatively different dynamics, which can be discriminated by neutron scattering. The methods of this paper are illustrated with oil/water microemulsion studied by small-angle scattering and neutron spin-echo. All available data-in both film and bulk contrasts, over the entire range of q and τ-are analyzed jointly with a single model. The analysis points to the static large-scale structure of the oil and water domains while the interfaces are subject to thermal fluctuations. The fluctuations have an amplitude of around 60 Å and contribute to 30% of the total interface area.

Mobility of bound water in PNIPAM microgels.

Polymer-solvent interactions play a crucial role in the stimuli-responsive behaviour of polymer networks. They influence the swelling/deswelling behaviour as well as the dynamics of the polymer chains. Scattering experiments provide insight into the polymer-water interaction of poly(N-isopropylacrylamide) (PNIPAM) microgels cross-linked with N,N'-methylenebisacrylamide (BIS) in dried and humidified state. The water mobility is studied by means of neutron spin-echo spectroscopy and neutron backscattering spectroscopy. The residual water amount has been determined with Karl Fischer titration. For both degrees of humidification, the relaxation time of the water molecules is much larger than that of free water due to the strong interactions with the polymer network and is only weakly depending on temperature and length scale of observation. The possible influence of the water on methyl group rotations is discussed.

Understanding near-surface polymer dynamics by a combination of grazing-incidence neutron scattering and virtual experiments.

Neutron spin-echo spectroscopy is a unique experimental method for the investigation of polymer dynamics. The combination of neutron spin-echo spectroscopy with grazing-incidence geometry (GINSES) opens the possibility to probe the dynamics of soft-matter materials in the vicinity of the solid substrate in the time range up to 100 ns. However, the usage of the GINSES technique has some peculiarities and, due to the novelty of the method and complexity of the scattering geometry, difficulties in further data analysis occur. The current work discusses how virtual experiments within the distorted-wave Born approximation using the BornAgain software can improve GINSES data treatment and aid the understanding of polymer dynamics in the vicinity of the solid surface. With two examples, poly(N-isopropyl acrylamide) brushes and poly(ethylene glycol) microgels on Si surfaces, the simulation as well as the application of the simulation to the GINSES data analysis are presented. The approach allowed a deeper insight to be gained of the background effect and scattering contribution of different layers.

Co-nonsolvency in concentrated aqueous solutions of PNIPAM: effect of methanol on the collective and the chain dynamics.

The polymer dynamics in concentrated solutions of poly(N-isopropyl acrylamide) (PNIPAM) in D2O/CD3OD mixtures is investigated in the one-phase region. Two polymer concentrations (9 and 25 wt%) and CD3OD contents in the solvent mixture of 0, 10 and 15 vol% are chosen. Temperature-resolved dynamic light scattering (DLS) reveals the collective dynamics. Two modes are observed, namely the fast relaxation of polymer segments within the blobs and the slow collective relaxation of the blobs. As the cloud point is approached, the correlation length related to the fast mode increases with CD3OD content. It features critical scaling behavior, which is consistent with mean-field behavior for the 9 wt% PNIPAM solution in pure D2O and with 3D Ising behavior for all other solutions. While the slow mode is not very strong in the 9 wt% PNIPAM solution in pure D2O, it is significantly more prominent as CD3OD is added and at all CD3OD contents in the 25 wt% solution, which may be attributed to enhanced interaction between the polymers. Neutron spin-echo spectroscopy (NSE) reveals a decay in the intermediate structure factor which indicates a diffusive process. For the polymer concentration of 9 wt%, the diffusion coefficients from NSE are similar to the ones from the fast relaxation observed in DLS. In contrast, they are significantly lower for the solutions having a polymer concentration of 25 wt%, which is attributed to the influence of the dominant large-scale dynamic heterogeneities. To summarize, addition of cosolvent leads to enhanced large-scale heterogeneities, which are reflected in the dynamic behavior at small length scales.

Effect of mild nanoscopic confinement on the dynamics of ionic liquids.

Ionic liquids are molten salts without an additional solvent and are discussed as innovative solvents and electrolytes in chemical processing and electrochemistry. A thorough microscopic understanding of the structure and ionic transport processes is essential for tailored applications. Here, we study the influence of "mild" nanoscopic confinement on the structure and diffusion properties of an ionic liquid, 1-ethyl-3-methylimidazolium acetate, using scattering techniques. The structure is analyzed by X-ray diffraction, while neutron backscattering spectroscopy is used for the study of the diffusion processes in these systems. Interpreting the diffusion processes in terms of a jump-diffusion model allowed us to deduce the confinement effects on the jump length and residence time, both increased at elevated temperatures in confinement. The applied "mild" confinement, which leaves room for 10-25 times the domain spacing, allows us to observe in great detail how the onset of domain distortion decelerates the dynamics.

Fuel Cell Electrode Characterization Using Neutron Scattering.

Electrochemical energy conversion and storage is key for the use of regenerative energies at large scale. A thorough understanding of the individual components, such as the ion conducting membrane and the electrode layers, can be obtained with scattering techniques on atomic to molecular length scales. The largely heterogeneous electrode layers of High-Temperature Polymer Electrolyte Fuel Cells are studied in this work with small- and wide-angle neutron scattering at the same time with the iMATERIA diffractometer at the spallation neutron source at J-PARC, opening a view on structural properties on atomic to mesoscopic length scales. Recent results on the proton mobility from the same samples measured with backscattering spectroscopy are put into relation with the structural findings.

Complex molecular dynamics of a symmetric model discotic liquid crystal revealed by broadband dielectric, thermal and neutron spectroscopy.

The molecular dynamics of the triphenylene-based discotic liquid crystal HAT6 is investigated by broadband dielectric spectroscopy, advanced dynamical calorimetry and neutron scattering. Differential scanning calorimetry in combination with X-ray scattering reveals that HAT6 has a plastic crystalline phase at low temperatures, a hexagonally ordered liquid crystalline phase at higher temperatures and undergoes a clearing transition at even higher temperatures. The dielectric spectra show several relaxation processes: a localized γ-relaxation at lower temperatures and a so called α2-relaxation at higher temperatures. The relaxation rates of the α2-relaxation have a complex temperature dependence and bear similarities to a dynamic glass transition. The relaxation rates estimated by Hyper DSC, Fast Scanning calorimetry and AC Chip calorimetry have a different temperature dependence than the dielectric α2-relaxation and follow the VFT-behavior characteristic for glassy dynamics. Therefore, this process is called α1-relaxation. Its relaxation rates show a similarity with that of polyethylene. For this reason, the α1-relaxation is assigned to the dynamic glass transition of the alkyl chains in the intercolumnar space. Moreover, this process is not observed by dielectric spectroscopy, which supports its assignment. The α2-relaxation is assigned to small scale translatorial and/or small angle fluctuations of the cores. The neutron scattering data reveal two relaxation processes. The process observed at shorter relaxation times is assigned to the methyl group rotation. The second relaxation process at longer time scales agree in the temperature dependence of its relaxation rates with that of the dielectric γ-relaxation.

Transition between protein-like and polymer-like dynamic behavior: Internal friction in unfolded apomyoglobin depends on denaturing conditions.

Equilibrium dynamics of different folding intermediates and denatured states is strongly connected to the exploration of the conformational space on the nanosecond time scale and might have implications in understanding protein folding. For the first time, the same protein system apomyoglobin has been investigated using neutron spin-echo spectroscopy in different states: native-like, partially folded (molten globule) and completely unfolded, following two different unfolding paths: using acid or guanidinium chloride (GdmCl). While the internal dynamics of the native-like state can be understood using normal mode analysis based on high resolution structural information of myoglobin, for the unfolded and even for the molten globule states, models from polymer science are employed. The Zimm model accurately describes the slowly-relaxing, expanded GdmCl-denaturated state, ignoring the individuality of the different aminoacid side chain. The dynamics of the acid unfolded and molten globule state are similar in the framework of the Zimm model with internal friction, where the chains still interact and hinder each other: the first Zimm relaxation time is as large as the internal friction time. Transient formation of secondary structure elements in the acid unfolded and presence of α-helices in the molten globule state lead to internal friction to a similar extent.

Surfactant Monolayer Bending Elasticity in Lipase Containing Bicontinuous Microemulsions.

Lipase-catalyzed reactions offer many advantages among which a high degree of selectivity combined with the possibility to convert even non-natural substrates are of particular interest. A major drawback in the applicability of lipases in the conversion of synthetically interesting, non-natural substrates is the substantial insolubility of such substrates in water. The conversion of substrates, natural or non-natural, by lipases generally involves the presence of a water-oil interface. In the present paper, we exploit the fact that the presence of lipases, in particular the lipase from Candida antarctica B (CalB), changes the bending elastic properties of a surfactant monolayer in a bicontinuous microemulsion consisting of D2O/NaCl -n-(d)-octane-pentaethylene glycol monodecyl ether (C10E5) in a similar manner as previously observed for amphiphilic block-copolymers. To determine the bending elastic constant, we have used two approaches, small angle neutron scattering (SANS) and neutron spin echo (NSE) spectroscopy. The time-averaged structure from SANS showed a slight decrease in bending elasticity, while on nanosecond time scales as probed with NSE, a stiffening has been observed, which was attributed to adsorption/desorption mechanisms of CalB at the surfactant monolayer. The results allow to derive further information on the influence of CalB on the composition and bending elasticity of the surfactant monolayer itself as well as the underlying adsorption/desorption mechanism.

Long-range excitations in phospholipid membranes.

We investigated the existence of long-range excitations and correlated structures in phospholipid membranes by means of grazing-incidence neutron spin echo spectroscopy, grazing-incidence small-angle neutron scattering, and corresponding theoretical calculations inspired by smectic-membrane theory. All these methods confirmed the existence of thermal excitations in the plane of the surface of the phospholipid membranes or the corresponding structures, respectively. Also, these measurements revealed a temperature dependence of these excitations. These excitations are associated with 100 nm in-plane correlations around physiological temperatures and of 75 nm at 16 °C. A single excitation has an energy around the µeV-regime. A temperature series revealed a high abundance at physiological temperatures and pronounced long-range in-plane structures, which are strongly suppressed at temperatures below 20 °C. From the length-scales and energy transfers involved we surmise that these excitations may play a role in several functions of the cell membranes such as stability and energy dissipation along the membrane. From a fundamental point of view, the observed behavior of those excitations is congruent with that of a quasi-particle (surface mode phonon, smomon) that exists in the plane of phospholipid membranes.

Inner structure and dynamics of microgels with low and medium crosslinker content prepared via surfactant-free precipitation polymerization and continuous monomer feeding approach.

The preparation of poly(N-isopropylacrylamide) microgels via classical precipitation polymerization (batch method) and a continuous monomer feeding approach (feeding method) leads to different internal crosslinker distributions, i.e., from core-shell-like to a more homogeneous one. The internal structure and dynamics of these microgels with low and medium crosslinker concentrations are studied with dynamic light scattering and small-angle neutron scattering in a wide q-range below and above the volume phase transition temperature. The influence of the preparation method, and crosslinker and initiator concentration on the internal structure of the microgels is investigated. In contrast to the classical conception where polymer microgels possess a core-shell structure with the averaged internal polymer density distribution within the core part, a detailed view of the internal inhomogeneities of the PNIPAM microgels and the presence of internal domains even above the volume phase transition temperature, when polymer microgels are in the deswollen state, are presented. The correlation between initiator concentration and the size of internal domains that appear inside the microgel with temperature increase is demonstrated. Moreover, the influence of internal inhomogeneities on the dynamics of the batch- and feeding-microgels studied with neutron spin-echo spectroscopy is reported.

Localised contacts lead to nanosecond hinge motions in dimeric bovine serum albumin.

Domain motions in proteins are crucial for biological function. In the present manuscript, we present a neutron spin-echo spectroscopy (NSE) study of native bovine serum albumin (BSA) in solution. NSE allows to probe both global and internal dynamics of the BSA monomer and dimer equilibrium that is formed in solution. Using a model independent approach, we were able to identify an internal dynamic process in BSA that is visible in addition to global rigid-body diffusion of the BSA monomer and dimer mixture. The observed internal protein motion is characterised by a relaxation time of 43 ns. The overdamped Brownian oscillator was considered as an alternative analytical theory that was able to describe the internal process as first-order approximation. More detailed information on the physical nature of the internal protein motion was extracted from the q-dependent internal diffusion coefficients ΔDeff(q) that were detected by NSE in addition to global rigid-body translational and rotational diffusion. The ΔDeff(q) were interpreted using normal mode analysis based on the available crystal structures of the BSA monomer and dimer as structural test models. Normal mode analysis demonstrates that the observed internal dynamic process can be attributed to bending motion of the BSA dimer. The native BSA monomer does not show any internal dynamics on the time- and length-scales probed by NSE. An intermolecular disulphide bridge or a direct structural contact between the BSA monomers forms a localised link acting as a molecular hinge in the BSA dimer. The effect of that hinge on the observed motion of BSA in the used dimeric structural model is discussed in terms of normal modes in a molecular picture.

A comparison of the network structure and inner dynamics of homogeneously and heterogeneously crosslinked PNIPAM microgels with high crosslinker content.

Poly(N-isopropylacrylamide) microgel particles were prepared via a "classical" surfactant-free precipitation polymerization and a continuous monomer feeding approach. It is anticipated that this yields microgel particles with different internal structures, namely a dense core with a fluffy shell for the classical approach and a more even crosslink distribution in the case of the continuous monomer feeding approach. A thorough structural investigation of the resulting microgels with dynamic light scattering, atomic force microscopy and small angle neutron scattering was conducted and related to neutron spin echo spectroscopy data. In this way a link between structural and dynamic features of the internal polymer network was made.

Proton diffusion in the catalytic layer for high temperature polymer electrolyte fuel cells.

The present study focuses on quasielastic neutron scattering (QENS) of the proton dynamics in phosphoric acid (PA) inside the catalytic layer of high-temperature polymer electrolyte fuel cells (HT-PEFCs). The nanosecond proton dynamics is investigated on the local length scale around operating temperatures (300 K-430 K) using neutron backscattering spectroscopy. We have investigated the catalyst doped with different amounts of PA in order to understand the distribution of PA inside the layer. Three approaches are considered for the description of proton dynamics: the random jump diffusion model, distribution of diffusion constants and, finally, the trap model. Due to adsorption of the PA on the Pt particles the diffusion of protons in the catalytic layer is different in comparison to the bulk acid. The proton dynamics in the catalytic layer can be described by the random jump diffusion with traps. This diffusion is significantly slower than the diffusion of free PA; this also results in a lower conductivity, which is estimated from the obtained diffusion constant.