On the origin of optical rotation changes during the κ-carrageenan disorder-to-order transition.

It is well established that solutions of both polymeric and oligomeric κ-carrageenan exhibit a clear change in optical rotation (OR), in concert with gel-formation for polymeric samples, as the solution is cooled in the presence of certain ions. The canonical interpretation - that this OR change reflects a 'coil-to-helix transition' in single chains - has seemed unambiguous; the solution- or 'disordered'-state structure has ubiquitously been assumed to be a 'random coil', and the helical nature of carrageenan in the solid-state was settled in the 1970s. However, recent work has found that κ-carrageenan contains substantial helical secondary structure elements in the disordered-state, raising doubts over the validity of this interpretation. To investigate the origins of the OR, density-functional theory calculations were conducted using atomic models of κ-carrageenan oligomers. Changes were found to occur in the predicted OR owing purely to dimerization of chains, and - together with the additional effects of slight changes in conformation that occur when separated helical chains form double-helices - the predicted OR changes are qualitatively consistent with experimental results. These findings contribute to a growing body of evidence that the carrageenan 'disorder-to-order' transition is a cooperative process, and have further implications for the interpretation of OR changes demonstrated by macromolecules in general.

Molecular dynamics simulations and X-ray scattering show the κ-carrageenan disorder-to-order transition to be the formation of double-helices.

Recent molecular dynamics simulations, verified experimentally by solution-state x-ray scattering experiments, have found that κ-carrageenan chains contain helical secondary structure, akin to that found in the solid-state, even in aqueous solution. Furthermore, upon the addition of ions to single chains the simulations found no evidence that any conformational transitions take place. These findings challenge the long-held assumption that the so-called disorder-to-order transition in carrageenan systems involves a uni-molecular 'coil-to-helix transition'. Herein, the results of further molecular dynamics simulations undertaken using pairs of κ-carrageenan chains in 0.1 M NaI solutions are reported, and are validated experimentally using state-of-the-art solution-state WAXS experiments. From initially separated chains double-helices are shown to form, leading the authors to propose 'two single helices-to-stabilized double-helix' as a description of the molecular events taking place during the disorder-to-order transition.

X-ray scattering and molecular dynamics simulations reveal the secondary structure of κ-carrageenan in the solution state.

The solution state structure of κ-carrageenan is typically described as a 'random coil', to indicate a lack of defined secondary structure elements. From this starting point the assignment of an optical-rotation-detected change that follows the introduction of particular ions to such solutions as a 'coil-to-helix transition' seems unambiguous, and thus the canonical description of this important biopolymer's gelling behaviour was born. However, the notion that κ-carrageenan exists in solution as a random coil, devoid of secondary structure, has been questioned a number of times previously in the literature, particularly by the molecular modelling and NMR communities. Regrettably, there has been little desire to-date to address these largely overlooked studies or consider their implications for the nature of the so-called 'coil-to-helix transition'. Despite evidence to the contrary, the random-coil-paradigm has prevailed. Here, new data from synchrotron-enabled solution-state x-ray scattering experiments, combined with state-of-the-art atomistic molecular dynamics simulations, are used to show that the solution-state structure of κ-carrageenan in fact retains many of the helical motifs present in the solid-state, as inferred from fibre diffraction data. Furthermore, no evidence is found to suggest that single chains undergo any uni-molecular conformational transition upon the addition of ions. These findings once again challenge the paradigm that κ-carrageenan exists as a 'random coil' in the solution state, and thereby question the long held assumption that a uni-molecular 'coil-to-helix transition' precedes the dimerization of helices.

Anomalous diffusion driven by the redistribution of internal stresses.

This article explores the mathematical description of anomalous diffusion, driven not by thermal fluctuations but by internal stresses. A continuous time random walk framework is outlined in which the waiting times between displacements (jumps), generated by the dynamics of internal stresses, are described by the generalized Γ distribution. The associated generalized diffusion equation is then identified. The solution to this equation is obtained as an integral over an infinite series of Fox H functions. The probability density function is identified as initially non-Gaussian, while at longer timescales Gaussianity is recovered. Likewise, the second moment displays a transient nature, shifting between subdiffusive and diffusive character. The potential application of this mathematical description to the quaking observed in several soft-matter systems is discussed briefly.

A comparative study of tough hydrogen bonding dissipating hydrogels made with different network structures.

Hydrogels are excellent soft materials to interface with biological systems. Precise control and tunability of dissipative properties of gels are particularly interesting in tissue engineering applications. In this work, we produced hydrogels with tunable dissipative properties by photopolymerizing a second polymer within a preformed cross-linked hydrogel network of poly(acrylamide). We explored second networks made with different structures and capacity to hydrogen bond with the first network, namely linear poly(acrylic acid) and branched poly(tannic acid). Gels incorporating a second network made with poly(tannic acid) exhibited excellent stiffness (0.35 ± 0.035 MPa) and toughness (1.64 ± 0.26 MJ m-3) compared to the poly(acrylic acid) counterparts. We also demonstrate a strategy to fabricate hydrogels where the dissipation (loss modulus) can be tuned independently from the elasticity (storage modulus) suitable for cell culture applications. We anticipate that this modular design approach for producing hydrogels will have applications in tailored substrates for cell culture studies and in load bearing tissue engineering applications.

A tale of two pectins: Diverse fine structures can result from identical processive PME treatments on similar high DM substrates.

The effects of a processive pectin-methylesterase (PME) treatment on two different pectins, both possessing a high degree of methylesterification (DM), were investigated. While the starting samples were purportedly very similar in fine structure, the intermolecular DM distributions arising from their PME treatments were strikingly different. Herein, a simulation that illuminates the origin of this phenomenon is described. It is concluded that: (1) very different low-DM samples (with the same average DM) can be generated using the same processive PME, simply by a judicious choice of the high DM starting material; (2) observing the intermolecular DM distribution of the products of processive-PME-processing is an extremely sensitive discriminator of the fine structure of high DM starting materials; and (3) for PMEs with unknown action patterns the processive nature of the enzyme is most simply revealed by studying the changes it induces in the intermolecular DM distribution of very-highly-methylesterified homogalacturonans.

Versatile multi-functionalization of protein nanofibrils for biosensor applications.

Protein nanofibrils offer advantages over other nanostructures due to the ease in their self-assembly and the versatility of surface chemistry available. Yet, an efficient and general methodology for their post-assembly functionalization remains a significant challenge. We introduce a generic approach, based on biotinylation and thiolation, for the multi-functionalization of protein nanofibrils self-assembled from whey proteins. Biochemical characterization shows the effects of the functionalization onto the nanofibrils' surface, giving insights into the changes in surface chemistry of the nanostructures. We show how these methods can be used to decorate whey protein nanofibrils with several components such as fluorescent quantum dots, enzymes, and metal nanoparticles. A multi-functionalization approach is used, as a proof of principle, for the development of a glucose biosensor platform, where the protein nanofibrils act as nanoscaffolds for glucose oxidase. Biotinylation is used for enzyme attachment and thiolation for nanoscaffold anchoring onto a gold electrode surface. Characterization via cyclic voltammetry shows an increase in glucose-oxidase mediated current response due to thiol-metal interactions with the gold electrode. The presented approach for protein nanofibril multi-functionalization is novel and has the potential of being applied to other protein nanostructures with similar surface chemistry.

An exploration of the microrheological environment around the distal ileal villi and proximal colonic mucosa of the possum (Trichosurus vulpecula).

Multiple particle-tracking techniques were used to quantify the thermally driven motion of ensembles of naked polystyrene (0.5 µm diameter) microbeads in order to determine the microrheological characteristics around the gut mucosa. The microbeads were introduced into living ex vivo preparations of the wall of the terminal ileum and proximal colon of the brushtail possum (Trichosurus vulpecula). The fluid environment surrounding both the ileal villi and colonic mucosa was heterogeneous; probably comprising discrete viscoelastic regions suspended in a continuous Newtonian fluid of viscosity close to water. Neither the viscosity of the continuous phase, the elastic modulus (G') nor the sizes of viscoelastic regions varied significantly between areas within 20 µm and areas more than 20 µm from the villous mucosa nor from the tip to the sides of the villous mucosa. The viscosity of the continuous phase at distances further than 20 µm from the colonic mucosa was greater than that at the same distance from the ileal villous mucosa. Furthermore, the estimated sizes of viscoelastic regions were significantly greater in the colon than in the ileum. These findings validate the sensitivity of the method and call into question previous hypotheses that a contiguous layer of mucus envelops all intestinal mucosa and restricts diffusive mass transfer. Our findings suggest that, in the terminal ileum and colon at least, mixing and mass transfer are governed by more complex dynamics than were previously assumed, perhaps with gel filtration by viscoelastic regions that are suspended in a Newtonian fluid.

On the Sulfation Pattern of Polysaccharides in the Extracellular Matrix of Sheep with Chondrodysplasia.

OBJECTIVE: Chondroitin sulfate is the major sulfated polysaccharide attached to the core protein, aggrecan, in the hyaline cartilage matrix. Sulfation of the cartilage matrix polysaccharide is vital for normal matrix integrity and compressive stiffness of the tissue and is therefore crucial to normal cartilage formation and consequently to endochondral ossification. Several forms of chondrodysplasia, a condition resulting in clear macroscopic deficiencies in the mechanical properties of the cartilage and characterized by reduced levels of sulfate, have been identified in both human beings and animals. DESIGN: In this study, the authors used capillary electrophoresis to investigate the sulfation state of extracted chondroitin sulfate polymers. RESULTS: Significantly, cartilage from affected sheep had a lower ratio of the chondroitin-derived enzymatically liberated disaccharides Δdi-mono4S to Δdi-mono6S, demonstrating reduced levels of chondroitin 4-sulfate, but not chondroitin 6-sulfate, in chondrodysplastic sheep compared to age-matched controls at all ages measured. CONCLUSION: This supports the hypothesis that a difference in chondroitin sulfate disaccharides is detectable in affected newborn lambs prior to the development of lesions.

Effects of weaning and infection with Teladorsagia circumcincta on mucin carbohydrate profiles of early weaned lambs.

Differences in mucin glycosylation in milk-fed and early weaned lambs may influence susceptibility to parasitism, particularly the greater cellular content and higher sulphation of mucins in young and unweaned lambs. Weaning also reduced the percentage of Gal (p<0.05) in fundic mucin and galactosamine (GalN) (p<0.01) in duodenal mucin, but had no noticeable effect on fucosylation or sialylation. Four experimental groups of lambs were studied (n=3): (1) 3 days old; (2) 9 weeks old milk-fed; (3) 9 weeks old weaned at 3 weeks-of-age on to lucerne chaff and cereal-based pellets (4) 9 weeks old weaned and infected with 1000 Teladorsagia circumcincta L3 twice weekly for 5 weeks. Fundic and duodenal mucin monosaccharides were analysed chemically and fundic, antral and duodenal tissues were stained with lectins, periodic acid Schiff (PAS), Alcian blue/PAS (AB/PAS) and high iron diamine. Age-related maturation of mucin glycosylation was prominent in young lambs: reduced total fundic mucins and increasing fucosylation and decreasing sialylation and sulphation of all mucins, as well as changes in the types of linkages of Fuc and sialic acids. By 9 weeks-of-age, there were no longer sialylated mucins in fundic surface mucus cells, only neutral mucins, while in Brunner's glands, there was reduced sialylation and large amounts of neutral mucins. In the neonates, both fundic and duodenal tissues contained only small amounts of mucins terminating with alpha-1,2-linked Fuc, which became the principal linkage in 9 weeks old lambs. Duodenal mucins in 3 days old lambs contained both alpha-2,6- and alpha-2,3-linked sialic acids, whereas the alpha-2,3 linkage was not present in older lambs. Parasitism increased the percentage of galactose, but reduced total and neutral fundic mucins, as well as sulphation and sialylation. There was both decreased sialylation and sulphation in duodenal mucins. Although no change in fucosylation was apparent from chemical analysis, infection reduced lectin staining for alpha-1,2-linked fucose in antral and duodenal tissues and alpha-1,6- and alpha-1,3-linked fucose in the duodenum. These changes in fundic and duodenal mucins were similar to those previously seen on Day 28 p.i. after a single infection of 4-9 months old sheep with T. circumcincta larvae.

Monosaccharide composition of fundic and duodenal mucins in sheep infected with Haemonchus contortus or Teladorsagia circumcincta.

The effects on the monosaccharide composition of fundic and duodenal mucins of parasitism by Haemonchus contortus or Teladorsagia circumcincta were investigated in sheep at 4, 6 and 9 months of age. Infected sheep were euthanased at days 21 and 28 post-infection respectively, together with uninfected controls. Fundic and duodenal mucins were purified by gel filtration and CsCl density gradient centrifugation and monosaccharides were released by heating at 95 degrees C for 6h in 2M HCl. Thin-layer chromatography identified fucose, glucosamine, galactose and galactosamine in both mucins, as well as very small amounts of sialic acids. Neither N-acetylglucosamine nor N-acetylgalactosamine was present, as these are deacetylated during acid hydrolysis to glucosamine and galactosamine, respectively. Fucose, glucosamine, galactose and galactosamine were separated and quantified by High Performance Anion Exchange Chromatography on a CarboPac PA-20 column. Sialic acids were determined by the thiobarbiturate assay. Over the age range of 4-9 months, the principal changes in the monosaccharide composition of mucins in non-infected sheep were increasing fucosylation and decreasing sialylation, as observed in other mammals. In duodenal mucins, there was a statistically significant increase in fucosylation and a decrease in sialylation (p=0.043 and 0.014, respectively), while similar trends were seen in fundic mucins. Other modifications with age in sheep mucins were decreased acetylglucosamine (N-acetylglucosamine) in the fundus and galactosamine (N-acetylgalactosamine) in the duodenum. The effects of H. contortus and T. circumcincta infection on fundic mucin monosaccharide composition were not identical, although both parasites decreased fucosylation and sialylation. Both parasites caused the same effects on duodenal mucins, however, these differed from the changes in the fundus. H. contortus infection increased the proportions of glucosamine and galactose in fundic and duodenal mucins, respectively. Mucins from the fundus of H. contortus-infected sheep had similar monosaccharide profiles at all ages, but this was not the case for T. circumcincta, in which there were lesser changes on mucins in 9 months old sheep, apart from decreased sialylation. This may indicate immunity to T. circumcincta from previous exposure in the field. The effect on duodenal mucins was similar for the two infections (decreased sialic acids, fucose and N-acetylgalactosamine and increased galactose), suggesting it may result from the immune response to the presence of worms in the abomasum. Mucin profiles from organs more accessible than the gastrointestinal tract may be useful markers for the host immune response and identify resistant, resilient or susceptible individuals.

Rheo-NMR studies of the behavior of a nematic liquid crystal in a low-shear-rate regime: the transition from director alignment to reorientation.

Deuterium NMR spectroscopy has been used to study the director dynamics of the nematic liquid-crystal system cetyl trimethylammonium bromide/D2O under the action of applied viscous torques. Shear forces were applied using a custom-built Couette cell that was introduced into an NMR superconducting magnet, so that its rotational axis was parallel to the magnetic field direction, along which the liquid-crystal director originally aligned. Subsequently, the inner cylinder of the cell was rotated continuously at different rates using a stepper motor. The resulting time evolution and ultimate steady-state orientation of the director, governed by the competition between the applied viscous torque with elastic and magnetic terms, was measured via observed changes in the deuterium spectrum. Using a simple gearbox allowed unprecedented access to a low-shear-rate regime in which, above a threshold shear rate, the director of part of the sample was observed to reorient, while the remaining part still aligned with the magnetic field. Subsequent increases in the applied rotational rate were found to increase the relative proportion of the orienting fraction. Spatially resolved NMR spectra showed that the orienting and field-aligned fractions formed separated bands across the gap of the Couette cell, with director reorientation being initiated at the moving inner wall. The behavior was found to be consistent with the often ignored variation in velocity gradient manifest across the gap of a cylindrical cell, so that as the angular frequency of the inner cylinder was increased the radial location of the critical shear rate required for reorientation traversed the gap. Once the applied rotational rate was sufficient to reorient the director of the entire sample, the dependence of the exhibited steady-state orientation on the average applied shear rate was measured. These results could be fitted to an analytical solution of the force-balance equation, made tractable by the assumption that the elasticity term was of minor significance and could be ignored. Additionally, the use of a numerical solution of the full force-balance equation, which explicitly includes elasticity and secondary flow and additionally allows the time evolution of the director orientation to be calculated, was investigated.

Insights into the potential functionality of single-chain force-induced conformational transitions in polymer networks: implications for polysaccharide signaling in the plant cell wall.

The behavior of biopolymer networks comprised of clickable polysaccharide chains that can undergo force-induced conformational transitions was investigated during straining using a simulation technique. The simulation was carried out both using an affine deformation field and alternatively using Lees-Edwards boundary conditions as an example of a nonaffine case. In the affine situation the simulated stress-strain curves were found to be consistent with results obtained by evaluating the molecular force-extension curve at a single average extension and calculating the bulk modulus as an average over all possible orientations with respect to the deformation. While in all cases examined the macroscopic mechanical responses of networks of randomly oriented chains, consisting either of simple extensible wormlike chains or their clickable analogs, were found to be indistinguishable, the simulation additionally allowed the number of chains containing sugar rings in different conformational states to be monitored, and this was found to change significantly during straining. This supports the hypothesis that in networks of randomly oriented clickable polysaccharide chains, such conformational transitions could have biological significance as stress switches in signaling processes but that they are unlikely to affect the bulk rheological properties of tissue.

Direct measurements of interfacial interactions between pectin and kappa-casein and implications for the stabilisation of calcium-free casein micelle mimics.

Using Surface Plasmon Resonance (SPR) it has been shown that the fine structure of the anionic polysaccharide pectin strongly influences its interfacial interaction with a kappa-casein layer coated onto a gold surface (via a dextran linker) in the pH range 3.5-6.8, with the highest SPR signal being observed for pectin with the lowest charge density tested (a degree of methylesterification (DM) around 90%). Furthermore, the Brownian motions of kappa-casein coated polystyrene beads (used to provide calcium-free 'model casein micelles') were studied in pectin solutions using Diffusing Wave Spectroscopy (DWS) and microscopy, and were compared with measurements made on naked beads. At every pH value studied (with the exception of 3.5), bridging of the protein-covered probe particles was observed for pectins of both DM 28 and DM 78. However, no aggregated complexes were found in these model casein micelle systems when pectin of an unusually high DM was used (90%). It was hypothesised that having a limited number of binding regions of spatially limited extent maximises the number of chains binding to the protein layer (as found with the SPR measurement), encourages the formation of loops and trains, and additionally limits the potential for destabilisation via bridging.

Bio-inspired network optimization in soft materials--insights from the plant cell wall.

The dynamic-mechanical responses of ionotropic gels made from the biopolymer pectin have recently been investigated by microrheological experiments and found to exhibit behaviour indicative of semi-flexible polymer networks. In this work we investigate the gelling behaviour of pectin systems in which an enzyme (pectinmethylesterase, PME) is used to liberate ion-binding sites on initially inert polymers, while in the presence of ions. This is in contrast to the previous work, where it was the release of ions (rather than ion-binding groups) that was controlled and the polymers had pre-existing cross-linkable moieties. In stark contrast to the semi-flexible network paradigm of biological gels and the previous work on pectin, the gels studied herein exhibit the properties of chemically cross-linked networks of flexible polymers.

Investigation of the effects of fine structure on the nanomechanical properties of pectin.

Pectin is an important structural polysaccharide found in the cell walls of all land plants. While in detail its composition and its organization in muro are complex, it is predominantly a copolymer of galacturonic acid and its methylesterified counterpart. Previous single-molecule stretching studies carried out on a sparsely methylesterified pectin sample indicated the importance of force-induced conformational transitions of the pyranose ring during extension, and the possible biological role of such transitions was discussed. More heavily methylesterified samples are better biomimetic models of the polymeric components as found in the plant cell wall, in particular being less restricted by the shackles of the significant intermolecular interactions expected to constrain the behavior of bare galacturonic acid sequences. Density functional theory calculations revealed that upon extending galacturonic acid monomers, whether methylesterified or not, the initial ((4)C1) chair structure is transformed to a ((3)S5) skew boat and that subsequently upon further elongation, via an intermediate inverted skew boat ((5)S3), the inverted chair ((1)C4) is reached. Experimentally, the force-extension curve of highly methylesterified pectin was found to be solvent dependent in the same manner as the un-esterified sample, indicating that minimal changes in the strength of interring hydrogen bonding result from such a substitution, and finally, as only subtle changes in the force-extension behavior of pectin resulted from changes in the degree of methylesterification, previous speculations about the role of force-induced transformations in vivo are supported.

Microrheological studies reveal semiflexible networks in gels of a ubiquitous cell wall polysaccharide.

Microrheological measurements have been carried out on ionotropic gels made from an important cell wall polysaccharide, using diffusing wave spectroscopy and multiple particle tracking. These gels were formed by the interaction of calcium ions with negatively charged groups on the polymer backbone, which is a copolymer of charged and uncharged sugars, galacturonic acid, and its methylesterified analog, respectively. The results suggest that semiflexible networks are formed in these systems, with a low frequency, frequency independent storage modulus (G'>G''), and a high frequency scaling of both G' and G'' with omega3/4. The differences observed between gels obtained using polysaccharide samples with different amounts and patterns of the charged ion-binding groups could comfortably be accommodated within this theoretical framework, assuming that the elementary semiflexible elements of the network are filaments consisting of two polymer chains bridged with calcium. In particular, a sample that was engineered to possess a blockwise intramolecular distribution of calcium chelating moieties clearly exhibited the high frequency scaling of both moduli with omega3/4 across some three orders of magnitude, and the concentration dependences of the elastic modulus, at both high and low frequency, were found to follow power laws with predicted exponents. Furthermore, quantitative agreement of the moduli with theory was found for realistic estimates of the molecular parameters, suggesting that the physics of semiflexible networks is not only exploited by protein components of the cytoskeleton but also by polysaccharides in plant cell walls.

Model for stretching elastic biopolymers which exhibit conformational transformations.

We derive an expression that represents the physical behavior of a polysaccharide molecule as it is stretched from the entropic region, through one or more ring conformational transformations, into the Hookean regime. The model adapts existing models in order to accommodate one or more force-induced conformational transformations of the glycan rings and is based on the concept of equilibrium between the clicked (longer conformers) and unclicked states. This equilibrium is determined by the Gibbs energy difference between these two states which is perturbed in favor of the clicked states by the force applied to the molecule. The derived expression is used to generate force-extension curves for model polymers and can illustrate the effect of the Gibbs energy for each transformation on the shape of these curves. It is also used to fit the force-extension curves of polysaccharides to obtain the Gibbs energy differences between the conformers. Good agreement was found between this model and experimental data on carboxymethylamylose, dextran, alginate, and pectin.