Differences in Water Dynamics between the Hydrated Chitin and Hydrated Chitosan Determined by Quasi-Elastic Neutron Scattering.

Recently, it was reported that chitin and chitosan exhibited high-proton conductivity and function as an electrolyte in fuel cells. In particular, it is noteworthy that proton conductivity in the hydrated chitin becomes 30 times higher than that in the hydrated chitosan. Since higher proton conductivity is necessary for the fuel cell electrolyte, it is significantly important to clarify the key factor for the realization of higher proton conduction from a microscopic viewpoint for the future development of fuel cells. Therefore, we have measured proton dynamics in the hydrated chitin using quasi-elastic neutron scattering (QENS) from the microscopic viewpoint and compared the proton conduction mechanism between hydrated chitin and chitosan. QENS results exhibited that a part of hydrogen atoms and hydration water in chitin are mobile even at 238 K, and the mobile hydrogen atoms and their diffusion increase with increasing temperature. It was found that the diffusion constant of mobile protons is two times larger and that the residence time is two times faster in chitin than that in chitosan. In addition, it is revealed from the experimental results that the transition process of dissociable hydrogen atoms between chitin and chitosan is different. To realize proton conduction in the hydrated chitosan, the hydrogen atoms of the hydronium ions (H3O+) should be transferred to another hydration water. By contrast, in hydrated chitin, the hydrogen atoms can transfer directly to the proton acceptors of neighboring chitin. It is deduced that higher proton conductivity in the hydrated chitin compared with that in the hydrated chitosan is yielded by the difference of diffusion constant and the residence time by hydrogen-atom dynamics and the location and number of proton acceptors.

Side Chain Dynamics of Poly(norbornene)-g-Poly(propylene oxide) Bottlebrush Polymers.

The segmental dynamics of the side chains of poly(norbornene)-g-poly(propylene oxide) (PNB-g-PPO) bottlebrush polymer in comparison to PPO is studied by quasi-elastic neutron scattering. Having experimental time and length scale information simultaneously allows to extract spatial information in addition to relaxation time. Tethering one end of the PPO side chain onto a stiff PNB backbone slows down the segmental relaxation, over the length and time scales investigated. The power law dependence of the relaxation time on the momentum transfer, Q, indicates a more heterogeneous relaxation pattern for the bottlebrush polymer, whereas the linear PPO has less deviations from a homogenous relaxation. Similar conclusions can be drawn from the time dependent mean square displacement, 〈r2 (t)〉, and the non-Gaussian parameter, α2 (t). Herein, the bottlebrush polymer shows a more restricted dynamics, whereas the linear PPO reaches 〈r2 (t)〉∝t0.5 at the highest temperature. The deviations from Gaussian behavior are evident at the α2 (t). Both samples show a decaying α2 (t). The non-Gaussian parameter supports the results from the power law dependence of the relaxation times, with lower α2 (t) values for PPO compared to those for PNB-g-PPO, pointing to less deviations from Gaussian behavior.

Hydrogen Dynamics in Hydrated Chitosan by Quasi-Elastic Neutron Scattering.

Chitosan, an environmentally friendly and highly bio-producible material, is a potential proton-conducting electrolyte for use in fuel cells. Thus, to microscopically elucidate proton transport in hydrated chitosan, we employed the quasi-elastic neutron scattering (QENS) technique. QENS analysis showed that the hydration water, which was mobile even at 238 K, moved significantly more slowly than the bulk water, in addition to exhibiting jump diffusion. Furthermore, upon increasing the temperature from 238 to 283 K, the diffusion constant of water increased from 1.33 × 10-6 to 1.34 × 10-5 cm2/s. It was also found that a portion of the hydrogen atoms in chitosan undergo a jump-diffusion motion similar to that of the hydrogen present in water. Moreover, QENS analysis revealed that the activation energy for the jump-diffusion of hydrogen in chitosan and in the hydration water was 0.30 eV, which is close to the value of 0.38 eV obtained from the temperature-dependent proton conductivity results. Overall, it was deduced that a portion of the hydrogen atoms in chitosan dissociate and protonate the interacting hydration water, resulting in the chitosan exhibiting proton conductivity.

Data Collection for Dilute Protein Solutions via a Neutron Backscattering Spectrometer.

Understanding protein functions requires not only static but also dynamic structural information. Incoherent quasi-elastic neutron scattering (QENS), which utilizes the highly incoherent scattering ability of hydrogen, is a powerful technique for revealing the dynamics of proteins in deuterium oxide (D2O) buffer solutions. The background scattering of sample cells suitable for aqueous protein solution samples, conducted with a neutron backscattering spectrometer, was evaluated. It was found that the scattering intensity of an aluminum sample cell coated with boehmite using D2O was lower than that of a sample cell coated with regular water (H2O). The D2O-Boehmite coated cell was used for the QENS measurement of a 0.8 wt.% aqueous solution of an intrinsically disordered protein in an intrinsically disordered region of a helicase-associated endonuclease for a fork-structured type of DNA. The cell was inert against aqueous samples at 283-363 K. In addition, meticulous attention to cells with small individual weight differences and the positional reproducibility of the sample cell relative to the spectrometer neutron beam position enabled the accurate subtraction of the scattering profiles of the D2O buffer and the sample container. Consequently, high-quality information on protein dynamics could be extracted from dilute protein solutions.

Overall structure of fully assembled cyanobacterial KaiABC circadian clock complex by an integrated experimental-computational approach.

In the cyanobacterial circadian clock system, KaiA, KaiB and KaiC periodically assemble into a large complex. Here we determined the overall structure of their fully assembled complex by integrating experimental and computational approaches. Small-angle X-ray and inverse contrast matching small-angle neutron scatterings coupled with size-exclusion chromatography provided constraints to highlight the spatial arrangements of the N-terminal domains of KaiA, which were not resolved in the previous structural analyses. Computationally built 20 million structural models of the complex were screened out utilizing the constrains and then subjected to molecular dynamics simulations to examine their stabilities. The final model suggests that, despite large fluctuation of the KaiA N-terminal domains, their preferential positionings mask the hydrophobic surface of the KaiA C-terminal domains, hindering additional KaiA-KaiC interactions. Thus, our integrative approach provides a useful tool to resolve large complex structures harboring dynamically fluctuating domains.

Experimental Evidence of Slow Mode Water in the Vicinity of Poly(ethylene oxide) at Physiological Temperature.

In some synthetic polymers used for medical applications, hydration water in the vicinity of the polymer chains is known to play an important role in biocompatibility and is referred to as intermediate water. The crystallization of water below 0 °C observed during thermal analysis has been considered as evidence of the presence of intermediate water. However, the origin and physicochemical properties of intermediate water have not yet been elucidated. In this study, as a typical biocompatible polymer, poly(ethylene oxide) and its hydration water were investigated with the use of terahertz time-domain spectroscopy and quasi-elastic neutron scattering. The obtained results prove the existence of a significant amount of mobile water that interacts with the polymer chains even when the water content is low at physiological temperatures.

Local Dynamics of the Hydration Water and Poly(Methyl Methacrylate) Chains in PMMA Networks.

The dynamic behavior of water molecules and polymer chains in a hydrated poly(methyl methacrylate) (PMMA) matrix containing a small amount of water molecules was investigated. Water molecules have been widely recognized as plasticizers for activating the segmental motion of polymer chains owing to their ability to reduce the glass transition temperature. In this study, combined with judicious hydrogen/deuterium labeling, we conducted quasi-elastic neutron scattering (QENS) experiments on PMMA for its dry and hydrated states. Our results clearly indicate that the dynamics of hydrated polymer chains are accelerated, and that individual water molecules are slower than bulk water. It is therefore suggested that the hydration water affects the local motion of PMMA and activates the local relaxation process known as restricted rotation, which is widely accepted to be generally insensitive to changes in the microenvironment.

Structural changes in pH-responsive gelatin/hydroxypropyl methylcellulose phthalate blends aimed at drug-release systems.

The present study aimed to investigate the thermal- and pH-dependent gelation behavior of gelatin/HPMCP blends using ultraviolet (UV) spectrophotometry, viscoelasticity, and dynamic light scattering (DLS). We found that the release of lisinopril from gelatin/HPMCP gels can be inhibited at low pH. UV spectrophotometric analysis showed that pH had a significant effect on the transparency of aqueous HPMCP systems and gelatin/HPMCP gels. The viscoelastic patterns of gelatin/HPMCP at pH 4.6 considerably differed from those of gelatin/HPMCP at pH 5.2 and 6.0. DLS measurements showed that HPMCP molecules in low concentrations underwent strong aggregation below pH 4.8. Such HPMCP aggregation induces a physical barrier in the matrix structures of the gelatin/HPMCP gels, which inhibits the drug release at pH 1.2. This hydrogel delivery system using polymer blends of gelatin/HPMCP can be used in oral gel formulations with pH-responsive properties.

Conformational dynamics of a multidomain protein by neutron scattering and computational analysis.

The flexible conformations of a multidomain protein are responsible for its biological functions. Although MurD, a 47-kDa protein that consists of three domains, sequentially changes its domain conformation from an open form to a closed form through a semiclosed form in its enzymatic reaction, the domain dynamics in each conformation remains unclear. In this study, we verify the conformational dynamics of MurD in the corresponding three states (apo and ATP- and inhibitor-bound states) with a combination of small-angle x-ray and neutron scattering (SAXS and SANS), dynamic light scattering (DLS), neutron backscattering (NBS), neutron spin echo (NSE) spectroscopy, and molecular dynamics (MD) simulations. Applying principal component analysis of the MD trajectories, twisting and open-closed domain modes are identified as the major collective coordinates. The deviations of the experimental SAXS profiles from the theoretical calculations based on the known crystal structures become smaller in the ATP-bound state than in the apo state, and a further decrease is evident upon inhibitor binding. These results suggest that domain motions of the protein are suppressed step by step of each ligand binding. The DLS and NBS data yield collective and self-translational diffusion constants, respectively, and we used them to extract collective domain motions in nanometer and nanosecond scales from the NSE data. In the apo state, MurD shows both twisting and open-closed domain modes, whereas an ATP binding suppresses twisting domain motions, and a further reduction of open-closed mode is seen in the inhibitor-binding state. These observations are consistent with the structure modifications measured by the small-angle scattering as well as the MD simulations. Such changes in the domain dynamics associated with the sequential enzymatic reactions should be related to the affinity and reaction efficiency with a ligand that binds specifically to each reaction state.

A feasibility study of inverse contrast-matching small-angle neutron scattering method combined with size exclusion chromatography using antibody interactions as model systems.

Small-angle neutron scattering (SANS) and small- angle X-ray scattering (SAXS) are powerful techniques for the structural characterization of biomolecular complexes. In particular, SANS enables a selective observation of specific components in complexes by selective deuteration with contrast-matching techniques. In most cases, however, biomolecular interaction systems with heterogeneous oligomers often contain unfavorable aggregates and unbound species, hampering data interpretation. To overcome these problems, SAXS has been recently combined with size exclusion chromatography (SEC), which enables the isolation of the target complex in a multi-component system. By contrast, SEC-SANS is only at a preliminary stage. Hence, we herein perform a feasibility study of this method based on our newly developed inverse contrast-matching (iCM) SANS technique using antibody interactions as model systems. Immunoglobulin G (IgG) or its Fc fragment was mixed with 75% deuterated Fc-binding proteins, i.e. a mutated form of IgG-degrading enzyme of Streptococcus pyogenes and a soluble form of Fcγ receptor IIIb, and subjected to SEC-SANS as well as SEC-SAXS as reference. We successfully observe SANS from the non-deuterated IgG or Fc formed in complex with these binding partners, which were unobservable in terms of SANS in D2O, hence demonstrating the potential utility of the SEC-iCM-SANS approach.

Dynamics of proteins with different molecular structures under solution condition.

Incoherent quasielastic neutron scattering (iQENS) is a fascinating technique for investigating the internal dynamics of protein. However, low flux of neutron beam, low signal to noise ratio of QENS spectrometers and unavailability of well-established analyzing method have been obstacles for studying internal dynamics under physiological condition (in solution). The recent progress of neutron source and spectrometer provide the fine iQENS profile with high statistics and as well the progress of computational technique enable us to quantitatively reveal the internal dynamic from the obtained iQENS profile. The internal dynamics of two proteins, globular domain protein (GDP) and intrinsically disordered protein (IDP) in solution, were measured with the state-of-the art QENS spectrometer and then revealed with the newly developed analyzing method. It was clarified that the average relaxation rate of IDP was larger than that of GDP and the fraction of mobile H atoms of IDP was also much higher than that of GDP. Combined with the structural analysis and the calculation of solvent accessible surface area of amino acid residue, it was concluded that the internal dynamics were related to the highly solvent exposed amino acid residues depending upon protein's structure.

Quasielastic neutron scattering study of microscopic dynamics in polybutadiene reinforced with an unsaturated carboxylate.

We studied the dynamics of zinc diacrylate (ZDA) reinforced polybutadiene rubber (BR) (ZDA/BR) using the quasielastic neutron scattering technique to determine the effect of concentration of ZDA on polymer dynamics. First, we evaluated the temperature dependence of mean square displacements (〈u2〉) for ZDA/BR with different ZDA volume fractions. 〈u2〉 increased with temperature below 170 K, and we observed no significant ZDA volume fraction dependence. However, it increased more steeply above 170 K, and the value of 〈u2〉 was smaller for the samples with increasing ZDA fraction. To elucidate the origin of the decrease in 〈u2〉 with increasing ZDA content, dynamic scattering laws (S(Q,ω)) were analyzed. An increase in the elastic component, an increase in the mean relaxation time, and a broadening of distribution of relaxation time were observed with the increasing volume fraction of ZDA. In addition, the ZDA volume fraction dependence of the elastic component roughly corresponded to that of elastic modulus, indicating that the elastic component is related to its mechanical strength. Referring to the previously reported static structure of the present ZDA/BR system, a model for the heterogeneous BR dynamics was proposed. This model assumes the coexistence of immobile, mobile, and interfacial constrained mobile regions. It was found to be appropriate for the explanation of the observed dynamics. We proposed that a network-like structure of the BR having a high crosslinking density around ZDA aggregates is mainly responsible for the high elastic modulus of ZDA/BR.

Modulation of the picosecond dynamics of troponin by the cardiomyopathy-causing mutation K247R of troponin T observed by quasielastic neutron scattering.

Troponin (Tn), consisting of three subunits (TnC, TnI, and TnT), regulates cardiac muscle contraction in a Ca2+-dependent manner. Various point mutations of human cardiac Tn are known to cause familial hypertrophic cardiomyopathy due to aberration of the regulatory function. In this study, we investigated the effects of one of these mutations, K247R of TnT, on the picosecond dynamics of the Tn core domain (Tn-CD), consisting of TnC, TnI and TnT2 (183-288 residues of TnT), by carrying out the quasielastic neutron scattering measurements on the reconstituted Tn-CD containing either the wild-type TnT2 (wtTn-CD) or the mutant TnT2 (K247R-Tn-CD) in the absence and presence of Ca2+. It was found that Ca2+-binding to the wtTn-CD decreases the residence time of atomic motions in the Tn-CD with slight changes in amplitudes, suggesting that the regulatory function mainly requires modulation of frequency of atomic motions. On the other hand, the K247R-Tn-CD shows different dynamic behavior from that of the wtTn-CD both in the absence and presence of Ca2+. In particular, the K247R-Tn-CD exhibits a larger amplitude than the wtTn-CD in the presence of Ca2+, suggesting that the mutant can explore larger conformational space than the wild-type. This increased flexibility should be relevant to the functional aberration of this mutant.

Dynamical Behavior of Hydration Water Molecules between Phospholipid Membranes.

The dynamical behavior of hydration water sandwiched between 1,2-dimyristyl-sn-glycero-3-phosphocholine (DMPC) bilayers was investigated by quasi-elastic neutron scattering (QENS) in the range between 275 and 316 K, where the main transition temperature of DMPC is interposed. The results revealed that the hydration water could be categorized into three types of water: (1) free water, whose dynamical behavior is slightly different from that of bulk water; (2) loosely bound water, whose dynamical behavior is 1 order of magnitude slower than that of the free water; and (3) tightly bound water, whose dynamical behavior is comparable with that of DMPC molecules. The number of loosely bound and tightly bound water molecules per DMPC molecule monotonically decreased and increased with decreasing temperature, respectively, and the sum of these water molecules remained constant. The number of free water molecules per DMPC molecule was constant in the measured temperature range.

Ligation-Dependent Picosecond Dynamics in Human Hemoglobin As Revealed by Quasielastic Neutron Scattering.

Hemoglobin, the vital O2 carrier in red blood cells, has long served as a classic example of an allosteric protein. Although high-resolution X-ray structural models are currently available for both the deoxy tense (T) and fully liganded relaxed (R) states of hemoglobin, much less is known about their dynamics, especially on the picosecond to subnanosecond time scales. Here, we investigate the picosecond dynamics of the deoxy and CO forms of human hemoglobin using quasielastic neutron scattering under near physiological conditions in order to extract the dynamics changes upon ligation. From the analysis of the global motions, we found that whereas the apparent diffusion coefficients of the deoxy form can be described by assuming translational and rotational diffusion of a rigid body, those of the CO form need to involve an additional contribution of internal large-scale motions. We also found that the local dynamics in the deoxy and CO forms are very similar in amplitude but are slightly lower in frequency in the former than in the latter. Our results reveal the presence of rapid large-scale motions in hemoglobin and further demonstrate that this internal mobility is governed allosterically by the ligation state of the heme group.

Hydrophilic Double-Network Polymers that Sustain High Mechanical Modulus under 80% Humidity.

The effects of water on the mechanical properties of synthetic hydrophilic polymers with double-network (DN) structures were studied under different relative humidities (RH). It was found that they could sustain nearly the same high Young's modulus as dry DN polymers in the RH range 10-80% (water content 3-17 wt %), that is, more than 102 MPa. However, when the RH exceeds 80%, DN polymers abruptly absorb large amounts of water (water content 90 wt %) and transform to a highly water-swollen "gel state" with a decrease in the Young's modulus of 3 orders of magnitude. Spectroscopic analyses revealed that water molecules below RH 80% are strongly bound to hydrophilic moieties with highly restricted mobility; water under such states improves rather than reduces mechanical properties by behaving as a plasticizer. DN polymers capable of sustaining high mechanical properties, even under RH 80%, have potential uses as hydrophilic materials.

Self-repairing filamentous actin hydrogel with hierarchical structure.

A chemically cross-linked filamentous actin (F-actin) gel consisting of globular actin (G-actin) as repeating units was prepared. The F-actin gel was cross-linked by covalent bonds, and the main chain is represented by the self-assembly of G-actin with a high-ordered hierarchical structure. The gel exhibited good mechanical performance with a storage modulus >1 kPa and undergoes reversible sol-gel transitions in response to changes in the salt concentration (chemical-induced sol-gel transition) as well as to shear strain (mechanical-induced sol-gel transition). Therefore, the gel exhibits self-repairing ability through dynamic polymerization and depolymerization across the structure hierarchies under repeated shear stress.

Surfactant-induced friction reduction for hydrogels in the boundary lubrication regime.

We studied the ability of surfactants to reduce friction by boundary lubrication for a bulk hydrogel sliding on a solid surface in an aqueous solution. A piece of negatively charged polyelectrolyte hydrogel was slid across solid surfaces with various levels of hydrophobicity, using a strain-controlled parallel-plate rheometer in water. A dramatic reduction in the sliding friction, especially in the low velocity region, was detected by the addition of a surfactant to the water medium. This friction reduction was only observed in gel-solid friction but not in solid-solid friction, indicating that the soft and wet nature of the gel surface was crucial for this surfactant-induced friction reduction. This phenomenon reveals that surfactants can remain at the gel-mated interface, thus preventing direct interfacial interaction between the sliding surfaces, and significantly decreasing the frictional stress. The reported dramatic reduction in friction highlights the frictional characteristics of soft and wet hydrogel materials.

Thermoresponsive microtubule hydrogel with high hierarchical structure.

A thermoresponsive 3D microtubule hydrogel (MT gel) was prepared by simultaneous polymerization and chemical cross-linking of tubulins. The main chain of this gel is composed of cross-linked MTs, which consists of a cylindrical assembly of tubulin covalently connected by polyethylene glycol. This gel, which contains 10 mg/mL of tubulin, exhibits a storage modulus G' as high as 1 × 10(3), which is 10 times higher than the loss modulus G'' over a wide range of frequencies. The MT gel exhibits a reversible sol-gel transition by temperature changes at 4-37 °C via depolymerization and polymerization of the MT network. Notable effects of the presence of the cross-linkage on the process of polymerization and depolymerization of tubulin were experimentally observed, and the role of the cross-linkage was discussed.

Interfacial water structure at polymer gel/quartz interfaces investigated by sum frequency generation spectroscopy.

Interfacial structures of water at polyvinyl alcohol (PVA) and poly(2-acrylamido-2-methypropane) sulfonic acid sodium salt (PNaAMPS)/quartz interfaces were investigated by sum frequency generation (SFG) spectroscopy. Two broad peaks were observed in OH stretching region at 3200 and 3400 cm(-1), corresponding to the symmetric OH stretching of tetrahedrally coordinated, i.e., strongly hydrogen bonded "ice-like" water, and the asymmetric OH stretching of water in a more random arrangement, i.e., weakly hydrogen bonded "liquid-like" water, respectively, in both cases. The "liquid-like" water became dominant when the PVA gel was pressed against the quartz surface. The relative intensity of the SFG signal due to the "liquid-like" water to that due to the "ice-like water" at the quartz surface modified with a self-assembled monolayer of aminopropyltrimethoxysilane (APS) became higher when the negatively charged PNaMPS gel was contacted to the APS modified quartz surface in a solution of pH = 12, where the surface was negatively charged and electrostatic repulsive interaction and low friction were present between the PNaMPS gel and the APS modified surface. It, however, did not change in a solution of pH = 2, where the surface was positively charged and electrostatic attractive interaction and very high friction were present between the PNaMPS gel and the APS modified surface. These results suggest the important role of water structure for small friction at the polymer gel/solid interface.