Detailed structural characterization of five water-insoluble α-glucans produced by glucansucrases from Streptococcus spp.

Water-insoluble α-glucans synthesized from sucrose by glucansucrases from Streptococcus spp. are essential in dental plaque and caries formation. Because limited information is available on the fine structure of these biopolymers, we analyzed the structures of unmodified glucans produced by five recombinant Streptococcus (S.) mutans DSM 20523 and S. salivarius DSM 20560 glucansucrases in detail. A combination of methylation analysis, endo-dextranase and endo-mutanase hydrolyses, and HPSEC-RI was used. Furthermore, crystal-like regions were analyzed by using XRD and 13C MAS NMR spectroscopy. Our results showed that the glucan structures were highly diverse: Two glucans with 1,3- and 1,6-linkages were characterized in detail besides an almost exclusively 1,3-linked and a linear 1,6-linked glucan. Furthermore, one glucan contained 1,3-, 1,4-, and 1,6-linkages and thus had an unusual, not yet described structure. It was demonstrated that the glucans had a varying structural architecture by using partial enzymatic hydrolyses. Furthermore, crystal-like regions formed by 1,3-glucopyranose units were observed for the two 1,3- and 1,6-linked glucans and the linear 1,3-linked glucan. 1,6-linked regions were mobile and not involved in the crystal-like areas. Altogether, our results broaden the knowledge of the structure of water-insoluble α-glucans from Streptococcus spp.

Disulfide-Cross-Linked Tetra-PEG Gels.

The preparation of polymer gels via cross-linking of four-arm star-shaped poly(ethylene glycol) (Tetra-PEG) precursors is an attractive strategy to prepare networks with relatively well-defined topologies. Typically, Tetra-PEG gels are obtained by cross-linking heterocomplementary reactive Tetra-PEG precursors. This study, in contrast, explores the cross-linking of self-reactive, thiol-end functional Tetra-PEG macromers to form disulfide-cross-linked gels. The structure of the disulfide-cross-linked Tetra-PEG gels was studied with multiple-quantum NMR (MQ-NMR) spectroscopy and small-angle neutron scattering (SANS) experiments. In line with earlier simulation studies, these experiments showed a strong dependence of the relative fractions of the different network connectivities on the concentration of the thiol-end functional Tetra-PEG macromer that was used for the synthesis of the networks. Disulfide-cross-linked Tetra-PEG gels prepared at macromer concentrations below the overlap concentration (c = 0.66c*) primarily feature defect connectivity motifs, such as primary loops and dangling ends. For networks prepared at macromer concentrations above the overlap concentration, the fraction of single-link connectivities was found to be similar to that in amide-cross-linked Tetra-PEG gels obtained by heterocomplementary cross-linking of N-hydroxysuccinimide ester and amine functional Tetra-PEG macromers. Since disulfide bonds are susceptible to reductive cleavage, these disulfide-cross-linked gels are of interest, e.g., as reduction-sensitive hydrogels for a variety of biomedical applications.

Time-domain proton-detected local-field NMR for molecular structure determination in complex lipid membranes.

Proton-detected local-field (PDLF) NMR spectroscopy, using magic-angle spinning and dipolar recoupling, is presently the most powerful experimental technique for obtaining atomistic structural information from small molecules undergoing anisotropic motion. Common examples include peptides, drugs, or lipids in model membranes and molecules that form liquid crystals. The measurements on complex systems are however compromised by the larger number of transients required. Retaining sufficient spectral quality in the direct dimension requires that the indirect time-domain modulation becomes too short for yielding dipolar splittings in the frequency domain. In such cases, the dipolar couplings can be obtained by fitting the experimental data; however ideal models often fail to fit PDLF data properly due to effects of radiofrequency field (RF) spatial inhomogeneity. Here, we demonstrate that by accounting for RF spatial inhomogeneity in the modeling of R-symmetry-based PDLF NMR experiments, the fitting accuracy is improved, facilitating the analysis of the experimental data. In comparison to the analysis of dipolar splittings without any fitting procedure, the accurate modeling of PDLF measurements makes possible three important improvements: the use of shorter experiments that enable the investigation of samples with a higher level of complexity, the measurement of C-H bond order parameters with smaller magnitudes |SCH| and of smaller variations of |SCH| caused by perturbations of the system, and the determination of |SCH| values with small differences from distinct sites having the same chemical shift. The increase in fitting accuracy is demonstrated by comparison with 2H NMR quadrupolar echo experiments on mixtures of deuterated and non-deuterated dimyristoylphosphatidylcholine (DMPC) and with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) membranes. Accurate modeling of PDLF NMR experiments is highly useful for investigating complex membrane systems. This is exemplified by application of the proposed fitting procedure for the characterization of membranes composed of a brain lipid extract with many distinct lipid types.

How entanglements determine the morphology of semicrystalline polymers.

Crystallization of polymers from entangled melts generally leads to the formation of semicrystalline materials with a nanoscopic morphology consisting of stacks of alternating crystalline and amorphous layers. The factors controlling the thickness of the crystalline layers are well studied; however, there is no quantitative understanding of the thickness of the amorphous layers. We elucidate the effect of entanglements on the semicrystalline morphology by the use of a series of model blends of high-molecular-weight polymers with unentangled oligomers leading to a reduced entanglement density in the melt as characterized by rheological measurements. Small-angle X-ray scattering experiments after isothermal crystallization reveal a reduced thickness of the amorphous layers, while the crystal thickness remains largely unaffected. We introduce a simple, yet quantitative model without adjustable parameters, according to which the measured thickness of the amorphous layers adjusts itself in such a way that the entanglement concentration reaches a specific maximum value. Furthermore, our model suggests an explanation for the large supercooling that is typically required for crystallization of polymers if entanglements cannot be dissolved during crystallization.

Unraveling an Alternative Mechanism in Polymer Self-Assemblies: An Order-Order Transition with Unusual Molecular Interactions between Hydrophilic and Hydrophobic Polymer Blocks.

Polymer self-assembly leading to cooling-induced hydrogel formation is relatively rare for synthetic polymers and typically relies on H-bonding between repeat units. Here, we describe a non-H-bonding mechanism for a cooling-induced reversible order-order (sphere-to-worm) transition and related thermogelation of solutions of polymer self-assemblies. A multitude of complementary analytical tools allowed us to reveal that a significant fraction of the hydrophobic and hydrophilic repeat units of the underlying block copolymer is in close proximity in the gel state. This unusual interaction between hydrophilic and hydrophobic blocks reduces the mobility of the hydrophilic block significantly by condensing the hydrophilic block onto the hydrophobic micelle core, thereby affecting the micelle packing parameter. This triggers the order-order transition from well-defined spherical micelles to long worm-like micelles, which ultimately results in the inverse thermogelation. Molecular dynamics modeling indicates that this unexpected condensation of the hydrophilic corona onto the hydrophobic core is due to particular interactions between amide groups in the hydrophilic repeat units and phenyl rings in the hydrophobic ones. Consequently, changes in the structure of the hydrophilic blocks affecting the strength of the interaction could be used to control macromolecular self-assembly, thus allowing for the tuning of gel characteristics such as strength, persistence, and gelation kinetics. We believe that this mechanism might be a relevant interaction pattern for other polymeric materials as well as their interaction in and with biological environments. For example, controlling the gel characteristics could be considered important for applications in drug delivery or biofabrication.

Early Stage UV-B Induced Molecular Modifications of Human Eye Lens γD-Crystallin.

In the human eye lenses, the crystallin proteins facilitate transparency, light refraction, as well as UV light protection. A deregulated balanced interplay between α-, ß-, and γ-crystallin can cause cataract. γD-crystallin (hγD) is involved in the energy dissipation of absorbed UV light by energy transfer between aromatic side chains. Early UV-B induced damage of hγD with molecular resolution is studied by solution NMR and fluorescence spectroscopy. hγD modifications are restricted to Tyr 17 and Tyr 29 in the N-terminal domain, where a local unfolding of the hydrophobic core is observed. None of the tryptophan residues assisting fluorescence energy transfer is modified and hγD is remained soluble over month. Investigating isotope-labeled hγD surrounded by eye lens extracts from cataract patients reveals very week interactions of solvent-exposed side chains in the C-terminal hγD domain and some remaining photoprotective properties of the extracts. Hereditary E107A hγD found in the eye lens core of infants developing cataract shows under the here used conditions a thermodynamic stability comparable to the wild type but an increased sensitivity toward UV-B irradiation.

Competition between crystal growth and intracrystalline chain diffusion determines the lamellar thickness in semicrystalline polymers.

The non-equilibrium thickness of lamellar crystals in semicrystalline polymers varies significantly between different polymer systems and depends on the crystallization temperature Tc. There is currently no consensus on the mechanism of thickness selection. Previous work has highlighted the decisive role of intracrystalline chain diffusion (ICD) in special cases, but a systematic dependence of lamellar thickness on relevant timescales such as that of ICD and stem attachment has not yet been established. Studying the morphology by small-angle X-ray scattering and the two timescales by NMR methods and polarization microscopy respectively, we here present data on poly(oxymethylene), a case with relatively slow ICD. It fills the gap between previously studied cases of absent and fast ICD, enabling us to establish a quantitative dependence of lamellar thickness on the competition between the noted timescales.

Defect-controlled softness, diffusive permeability, and mesh-topology of metallo-supramolecular hydrogels.

Hydrogels are polymer networks swollen in water; they are suitable materials for biomedical applications such as tissue engineering and drug delivery. In the latter, the controlled diffusion of small diffusants inside the network is essential, as it determines the release mechanism of the drug. In general, the diffusion inside a polymer network is controlled by its mesh-size. Here, we actively control the diffusivity and also the softness of metallo-supramolecular hydrogels via the network mesh-topology by introducing connectivity defects. A model polymer network is realized based on a 4-arm poly(ethylene glycol) (pEG) where each arm is capped with terpyridine moieties that are capable of forming metallo-supramolecular complexes with zinc ions. In this model network, we insert 8-arm pEG macromolecules that are functionalized with terpyridine at different ratios to create connectivity defects. With an increasing amount of 8-arm pEG, the polymer network forms more loops, as quantified by double quantum-NMR. This doped network shows an enhanced self-diffusivity of the building block molecules within the network, as examined by fluorescence recovery after photobleaching, and a higher softness, as investigated by oscillatory shear rheology. With these findings, we show that it is possible to tune the diffusivity and softness of hydrogels with defects in a rational fashion.

Polymer Networks for Enrichment of Calcium Ions.

In this study, solvogels containing (2-((2-(ethoxycarbonyl)prop-2-en-1-yl)oxy)-ethyl) phosphonic acid (ECPA) and N,N'-diethyl-1,3-bis-(acrylamido)propane (BNEAA) as the crosslinker are synthesized by UV induced crosslinking photopolymerization in various solvents. The polymerization of the ECPA monomer is monitored by the conversion of double bonds with in situ attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The morphology of the networks is characterized by in situ photorheology, solid state NMR spectroscopy, and scanning electron microscopy (SEM) of the dried gels. It is demonstrated that the storage modulus is not only determined by the crosslinker content in the gel, but also by the solvent used for preparation. The networks turn out to be porous structures with G' being governed by a rigid, phase-separated polymer phase rather than by entropic elasticity. The external and internal pKa values of the poly(ECPA-co-BNEAA) gels were determined by titration with a specially designed method and compared to the calculated values. The polymer-immobilized phosphonic acid groups in the hydrogels induce buffering behavior into the system without using a dissolved buffer. The calcium accumulation in the gels is studied by means of a double diffusion cell filled with calcium ion-containing solutions. The successful accumulation of hydroxyapatite within the gels is shown by a combination of SEM, energy-dispersive X-ray spectroscopy (EDX) and wide-angle X-ray scattering (WAXS).

Sulfobetaine Hydrogels with a Complex Multilength-Scale Hierarchical Structure.

Hydrogels with a hierarchical structure were prepared from a new highly water-soluble crosslinker N,N,N',N'-tetramethyl-N,N'-bis(2-ethylmethacrylate)-propyl-1,3-diammonium dibromide and from the sulfobetaine monomer 2-(N-3-sulfopropyl-N,N-dimethyl ammonium)ethyl methacrylate. The free radical polymerization of the two compounds is rapid and yields near-transparent hydrogels with sizes up to 5 cm in diameter. Rheology shows a clear correlation between the monomer-to-crosslinker ratio and the storage and loss moduli of the hydrogels. Cryo-scanning electron microscopy, low-field nuclear magnetic resonance (NMR) spectroscopy, and small-angle X-ray scattering show that the gels have a hierarchical structure with features spanning the nanometer to the sub-millimeter scale. The NMR study is challenged by the marked inhomogeneity of the gels and the complex chemical structure of the sulfobetaine monomer. NMR spectroscopy shows how these complications can be addressed via a novel fitting approach that considers the mobility gradient along the side chain of methacrylate-based monomers.

Dynamic Heterogeneity of Filler-Associated Interphases in Polymer Nanocomposites.

Dynamically inhomogeneous polymer systems exhibit interphases with mobility gradients. These are believed to play key roles in the material's performance. A prominent example is particle-filled rubber, a special case of a crosslinked polymer nanocomposite, where favorable rubber-filler interactions may give rise to a nanoscale immobilized layer around the filler, including regions of intermediate mobility. Such intermediate domains may either form a separate shell-like layer or be a manifestation of dynamic heterogeneities, in which case the intermediately mobile material would be dispersed in the form of nanometer-sized subdomains. In this contribution, bidirectional proton NMR spin diffusion (SD) experiments applied to silica-filled acrylate rubber are combined with numerical simulations to provide microscopic insights into this question. While model calculations for different scenarios fit the given data similarly well for longer SD mixing time, the short-time data do support the presence of dynamic heterogeneities.

Spatial inhomogeneity, interfaces and complex vitrification kinetics in a network forming nanocomposite.

A detailed calorimetric study on an epoxy-based nanocomposite system was performed employing bisphenol A diglycidyl ether (DGEBA) cured with diethylenetriamine (DETA) as the polymer matrix and a taurine-modified MgAL layered double hydroxide (T-LDH) as the nanofiller. The -NH2 group of taurine can react with DGEBA improving the interaction of the polymer with the filler. The combined X-ray scattering and electron microscopy data showed that the nanocomposite has a partially exfoliated morphology. Calorimetric studies were performed using conventional DSC, temperature modulated DSC (TMDSC) and fast scanning calorimetry (FSC) in the temperature modulated approach (TMFSC) to investigate the vitrification and molecular mobility dependent on the filler concentration. First, TMDSC and NMR were used to estimate the amount of the rigid amorphous fraction which consists of immobilized polymer segments at the nanoparticle surface. It was found to be 40 wt% for the highest filler concentration, indicating that the interface dominates the overall macroscopic properties and behavior of the material to a great extent. Second, the relaxation rates of the α-relaxation obtained by TMDSC and TMFSC were compared with the thermal and dielectric relaxation rates measured by static FSC. The investigation revealed that the system shows two distinct α-relaxation processes. Furthermore, two separate vitrification mechanisms were also found for a bulk network-former without geometrical confinement as also confirmed by NMR. This was discussed in terms of the intrinsic spatial heterogeneity on a molecular scale, which becomes more pronounced with increasing nanofiller content.

Efficient polynomial analysis of magic-angle spinning sidebands and application to order parameter determination in anisotropic samples.

Chemical shift tensors in 13C solid-state NMR provide valuable localized information on the chemical bonding environment in organic matter, and deviations from isotropic static-limit powder line shapes sensitively encode dynamic-averaging or orientation effects. Studies in 13C natural abundance require magic-angle spinning (MAS), where the analysis must thus focus on spinning sidebands. We propose an alternative fitting procedure for spinning sidebands based upon a polynomial expansion that is more efficient than the common numerical solution of the powder average. The approach plays out its advantages in the determination of CST (chemical-shift tensor) principal values from spinning-sideband intensities and order parameters in non-isotropic samples, which is here illustrated with the example of stretched glassy polycarbonate.

Terminal Flow of Cluster-Forming Supramolecular Polymer Networks: Single-Chain Relaxation or Micelle Reorganization?

We correlate the terminal relaxation of supramolecular polymer networks, based on unentangled telechelic poly(isobutylene) linear chains forming micellar end-group clusters, with the microscopic chain dynamics as probed by proton NMR. For a series of samples with increasing molecular weight, we find a quantitative agreement between the terminal relaxation times and their activation energies provided by rheology and NMR. This finding corroborates the validity of the transient-network model and the special case of the sticky Rouse model, and dismisses more dedicated approaches treating the terminal relaxation in terms of micellar rearrangements. Also, we confirm previous results showing reduction of the activation energy of supramolecular dissociation with increasing molecular weight and explain this trend with an increasing elastic penalty, as corroborated by small angle x-ray scattering data.

Relaxation-induced dipolar exchange with recoupling (RIDER) distortions in CODEX experiments.

Chemical shift anisotropy (CSA) and dipolar CODEX (Cenralband Only Detection of EXchange) experiments enable abundant quantitative information on the reorientation of the CSA and dipolar tensors to be obtained on millisecond-second timescales. At the same time, proper performance of the experiments and data analysis can often be a challenge since CODEX is prone to some interfering effects that may lead to incorrect interpretation of the experimental results. One of the most important such effects is RIDER (relaxation-induced dipolar exchange with recoupling). It appears due to the dipolar interaction of the observed X nuclei with some other nuclei, which causes an apparent decay in the mixing time dependence of the signal intensity reflecting not molecular motion, but spin flips of the adjacent nuclei. This may hamper obtaining correct values of the parameters of molecular mobility. In this contribution we consider in detail the reasons why the RIDER distortions remain even under decoupling conditions and propose measures to eliminate them. That is, we suggest (1) using an additional Z filter between the cross-polarization (CP) section and the CODEX recoupling blocks that suppresses the interfering anti-phase coherence responsible for the X-H RIDER and (2) recording only the cosine component of the CODEX signal since it is less prone to the RIDER distortions in comparison to the sine component. The experiments were conducted on rigid model substances as well as microcrystalline 2H / 15N-enriched proteins (GB1 and SH3) with a partial back-exchange of labile protons. Standard CSA and dipolar CODEX experiments reveal a fast-decaying component in the mixing time dependence of 15N nuclei in proteins, which can be misinterpreted as a slow overall protein rocking motion. However, the RIDER-free experimental setup provides flat mixing time dependences, meaning that the studied proteins do not undergo global motions on the millisecond timescale.

Initial Solvent-Driven Nonequilibrium Effect on Structure, Properties, and Dynamics of Polymer Nanocomposites.

Unusual structures and dynamic properties found in polymer nanocomposites (PNCs) are often attributed to immobilized (adsorbed) polymers at nanoparticle-polymer interfaces, which are responsible for reducing the intrinsic incompatibility between nanoparticles and polymers in PNCs. Although tremendous effort has been made to characterize the presence of immobilized polymers, a systematic understanding of the structure and dynamics under different processing conditions is still lacking. Here, we report that the initial dispersing solvent, which is not present after producing PNCs, drives these nonequilibrium effects on polymer chain dynamics at interfaces. Employing extensive small-angle scattering, proton NMR spectroscopy, and rheometry experiments, we found that the thickness of the immobilized layer can be dependent on the initial solvent, changing the structure and the properties of the PNC significantly. In addition, we show that the outcome of the initial solvent effect becomes more effective at particle volume fractions where the immobile layers begin to interact.

Quantitative NMR study of heat-induced aggregation of eye-lens crystallin proteins under crowding conditions.

The eye lens contains a highly concentrated, polydisperse mixture of crystallins, and a loss in transparency during cataract formation is attributed to the aggregation of these proteins. Most biochemical and biophysical studies of crystallins have been performed in diluted samples because of various physical limitations of the respective method at physiological concentrations of up to 200-400 mg/mL. We introduce a straightforward proton NMR transverse relaxometry method to quantify simultaneously proteins in the dissolved and aggregated states at these elevated concentrations, because these states significantly differ in their transverse relaxation properties. The key feature of this method is a direct observation of the protein signal in a wide range of relaxation delays, from few microseconds up to few hundred milliseconds. We applied this method to follow heat-induced aggregation of bovine α- and γB-crystallin between 60 and 200 mg/mL. We find that at 60 °C, a temperature where both crystallins still comprise a native tertiary structure, γB-crystallin aggregated at these high protein concentrations with a time constant of about 30-40 h. α-crystallin remained soluble at 60 mg/mL but formed a transparent gel at 200 mg/mL. This quantitative NMR method can be applied to investigations of other proteins and their mixtures under various aggregation conditions.

The Influence of Chemical Modification on Linker Rotational Dynamics in Metal-Organic Frameworks.

The robust synthetic flexibility of metal-organic frameworks (MOFs) offers a promising class of tailorable materials, for which the ability to tune specific physicochemical properties is highly desired. This is achievable only through a thorough description of the consequences for chemical manipulations both in structure and dynamics. Magic angle spinning solid-state NMR spectroscopy offers many modalities in this pursuit, particularly for dynamic studies. Herein, we employ a separated-local-field NMR approach to show how specific intraframework chemical modifications to MOF UiO-66 heavily modulate the dynamic evolution of the organic ring moiety over several orders of magnitude.

Microsecond motions probed by near-rotary-resonance R1ρ15N MAS NMR experiments: the model case of protein overall-rocking in crystals.

Solid-state near-rotary-resonance measurements of the spin-lattice relaxation rate in the rotating frame (R1ρ) is a powerful NMR technique for studying molecular dynamics in the microsecond time scale. The small difference between the spin-lock (SL) and magic-angle-spinning (MAS) frequencies allows sampling very slow motions, at the same time it brings up some methodological challenges. In this work, several issues affecting correct measurements and analysis of 15N R1ρ data are considered in detail. Among them are signal amplitude as a function of the difference between SL and MAS frequencies, "dead time" in the initial part of the relaxation decay caused by transient spin-dynamic oscillations, measurements under HORROR condition and proper treatment of the multi-exponential relaxation decays. The multiple 15N R1ρ measurements at different SL fields and temperatures have been conducted in 1D mode (i.e. without site-specific resolution) for a set of four different microcrystalline protein samples (GB1, SH3, MPD-ubiquitin and cubic-PEG-ubiquitin) to study the overall protein rocking in a crystal. While the amplitude of this motion varies very significantly, its correlation time for all four sample is practically the same, 30-50 µs. The amplitude of the rocking motion correlates with the packing density of a protein crystal. It has been suggested that the rocking motion is not diffusive but likely a jump-like dynamic process.