The Assembly Mechanism and Mesoscale Architecture of Protein-Polysaccharide Complexes Formed at the Solid-liquid Interface.

Protein-polysaccharide composite materials have generated much interest due to their potential use in medical science and biotechnology. A comprehensive understanding of the assembly mechanism and the mesoscale architecture is needed for fabricating protein-polysaccharide composite materials with desired properties. In this study, complex assemblies were built on silica surfaces through a layer-by-layer (LbL) approach using bovine beta-lactoglobulin variant A (ßLgA) and pectin as model protein and polysaccharide, respectively. We demonstrated the combined use of quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR) for elucidating the assembly mechanism as well as the internal architecture of the protein-polysaccharide complexes formed at the solid-liquid interface. Our results show that ßLgA and pectin interacted with each other and formed a cohesive matrix structure at the interface consisting of intertwined pectin chains that were cross-linked by ßLgA-rich domains. Although the complexes were fabricated in an LbL fashion, the complexes appeared to be relatively homogeneous with ßLgA and pectin molecules spatially distributed within the matrix structure. Our results also demonstrate that the density of ßLgA-pectin complex assemblies increased with both the overall and local charge density of pectin molecules. Therefore, the physical properties of the protein-polysaccharide matrix structure, including density and level of hydration, can be tuned by using polysaccharides with varying charge patterns, thus promoting the development of composite materials with desired properties.

Locking the Spiro Carbon in Spirobisindane Using Sulfur and Phosphorus to Form "Olympic Ring"-like Molecules.

To improve the rigidity of spirobisindane, it was intramolecularly locked by forming eight-membered rings via sulfur and phosphorus atoms to produce an interlocked polycyclic structure under mild conditions in good yields. By carefully analyzing the crystal structures, we noticed that the angle between the two benzene rings in the locked version is significantly smaller than that of the typical spirobisindane structure. Molecular modeling indicated that locking the spiro center can remarkably enhance the rigidity.

Geometric Frustration and Long-Range Ordering Induced by Surface Pressure Oscillation in a Langmuir-Blodgett Monolayer of Magnetic Soft Spheres.

As a step toward the bottom-up construction of magnonic systems, this paper demonstrates the use of a large-amplitude surface-pressure annealing technique to generate 2-D order in a Langmuir-Blodgett monolayer of magnetic soft spheres comprising a surfactant-encapsulated polyoxometalate. The films show a distorted square lattice interpreted as due to geometric frustration caused by 2-D confinement between soft walls, one being the air interface and the other the aqueous subphase. Hysteresis and relaxation phenomena in the 2-D layers are suggested to be due to folding and time-dependent interpenetration of surfactant chains.

Synthesis and characterization of mono S-lipidated peptide hydrogels: a platform for the preparation of reactive oxygen species responsive materials.

In this work we report the synthesis of mono lipidated peptides containing a 3-mercaptopropionate linker in the N-terminus by means of a photoinitiated thiol-ene reaction (S-lipidation). We evaluate the self-assembling and hydrogelation properties of a library of mono S-lipidated peptides containing lipid chains of various lengths and demonstrate that hydrogelation was driven by a balance between the lipid chain's hydrophobicity and the peptide's facial hydrophobicity. We further postulate that a simple calculation using estimated values of log D could be used as a predictor of hydrogelation when designing similar systems. A mono S-lipidated peptide containing a short lipid chain that formed hydrogels was fully characterized and a mechanism for the peptide hydrogelation developed. Finally, we demonstrate that the presence of the thioether group in the mono S-lipidated peptide hydrogels, which is a feature lacking in conventional N-acyl lipidated systems, enables the controlled disassembly of the gel via oxidation to the sulfoxide by reactive oxygen species in accordance with a hydrophobicity-modulated strategy. Thus, we conclude that mono S-lipidated peptide hydrogels constitute a novel and simple tool for the development of tissue engineering and targeted drug delivery applications of diseases with overexpression of reactive oxygen species (e.g. degenerative and metabolic diseases, and cancers).

Aligned Assembly in a 2-D Gel of a Water-Soluble Peptide.

We demonstrate the assembly of a compact, gel-like Langmuir-Blodgett film of rods formed by self-assembly of a ß-sheet-forming water-soluble peptide, Ac-IKHLSVN-NH2, at the surface of aqueous electrolytes. We characterize surface pressure hysteresis and demonstrate shear stiffening of the surface caused by area cycling, which we interpret as due to rearrangement and alignment of the rods. We show strong effects of the electrolyte on the assembly of the elementary rods, which can be related to the Hofmeister series and interpreted by effects on the interaction energies mediated by ions and water. Formation of ß-sheet structures and assembly of these into surface-segregated semicrystalline gels was strongly promoted by ammonium sulfate electrolyte. With ammonium sulfate electrolyte as subphase for Langmuir-Blodgett film deposition, shear stiffening by surface area cycling resulted in very compact films on transfer to a substrate.

Directed self-assembly of peptide-diketopyrrolopyrrole conjugates - a platform for bio-organic thin film preparation.

Increased water solubility and long-range intermolecular ordering have been introduced into the fluorescent organic molecule thiophene-diketopyrrolopyrrole (TDPP) via its conjugation to the octapeptide HEFISTAH, which is derived from the protein-protein ß-interface of the homo-tetramer protein diaminopimelate decarboxylase. The octapeptide, and its TDPP mono- and cross-linked conjugates were synthesised using 9-fluorenylmethoxycarbonyl (Fmoc) based solid-phase peptide synthesis (SPPS). Unlike the unmodified peptide, the resulting mono-linked and cross-linked peptides showed a fibrous morphology and formed hydrogels at 4 wt% in water at neutral pH, but failed to assemble at pH 2 and pH 9. Further peptide characterization showed that the TDPP organic core enhances peptide self-assembly and that both peptides assembled into fibers with a parallel ß-sheet structure. Furthermore, UV-vis spectroscopic analysis suggests that the TDPP molecules form H-type aggregates where the chromophores are likely to be co-facially packed, but rotationally and/or laterally offset from one another. This intermolecular coupling indicates that π-π stacking interactions are highly likely - a favourable sign for charge transport. The enhanced aqueous solubility and self-assembling properties of the TDPP-peptide conjugates allowed the successful preparation of thin films. Atomic force microscopy, X-ray diffraction and UV-vis spectroscopic analysis of these thin films revealed that the hybrid materials retained a fibrous morphology, ß-sheet structures and strong intermolecular coupling between neighbouring TDPP molecules. These results open an exciting avenue for bio-organic materials development, through structural and electronic tuning of the TDPP core.

Changes in the orientations of cellulose microfibrils during the development of collenchyma cell walls of celery (Apium graveolens L.).

MAIN CONCLUSION: During development, cellulose microfibrils in collenchyma walls become increasingly longitudinal, as determined by small-angle X-ray scattering, despite the walls maintaining a fine structure indicative of a crossed-polylamellate structure. Collenchyma cells have thickened primary cell walls and provide mechanical support during plant growth. During their development, these cells elongate and their walls thicken considerably. We used microscopy and synchrotron small-angle X-ray scattering to study changes in the orientations of cellulose microfibrils that occur during development in the walls of collenchyma cells present in peripheral strands in celery (Apium graveolens) petioles. Transmission electron microscopy showed that the walls consisted of many lamellae (polylamellate), with lamellae containing longitudinally oriented cellulose microfibrils alternating with microfibrils oriented at higher angles. The lamellae containing longitudinally oriented microfibrils predominated at later stages of development. Nevertheless, transmission electron microscopy of specially stained, oblique sections provided evidence that the cellulose microfibrils were ordered throughout development as crossed-polylamellate structures. These results are consistent with our synchrotron small-angle X-ray scattering results that showed the cellulose microfibrils become oriented increasingly longitudinally during development. Some passive reorientation of cellulose microfibrils may occur during development, but extensive reorientation throughout the wall would destroy ordered structures. Atomic force microscopy and field emission scanning electron microscopy were used to determine the orientations of newly deposited cellulose microfibrils. These were found to vary widely among different cells, which could be consistent with the formation of crossed-polylamellate structures. These newly deposited cellulose microfibrils are deposited in a layer of pectic polysaccharides that lies immediately outside the plasma membrane. Overall, our results show that during development of collenchyma walls, the cellulose microfibrils become increasingly longitudinal in orientation, yet organized, crossed-polylamellate structures are maintained.

Soft and Hard Interactions between Polystyrene Nanoplastics and Human Serum Albumin Protein Corona.

Upon contact with biological fluids, the surface of nanoparticles is surrounded by many types of proteins, forming a so-called "protein corona". The physicochemical properties of the nanoparticle/corona complex depend predominantly on the nature of the protein corona. An understanding of the structure of the corona and the resulting complex provides insight into the structure-activity relationship. Here, we structurally evaluate the soft and hard components of the protein corona, formed from polystyrene (PS) nanoplastics and human serum albumin (HSA). Using circular dichroism spectroscopy to elucidate the structure of HSA within the complex, we establish the effect of nanoparticle size and pH on the nature of the protein corona formed- whether hard or soft. Despite the weak interaction between PS and the HSA corona, small angle neutron scattering revealed the formation of a complex structure that enhanced the intermolecular interactions between HSA proteins, PS particles, and the HS/PSA complexes. Fractal formation occurred under conditions where the interaction between PS and HSA was strong, and increasing HSA concentrations suppressed the degree of aggregation. The size of the nanoparticles directly influenced the nature of the protein corona, with larger particles favoring the formation of a soft corona, due to the decreased PS-HSA attraction.

Solution structure of linear battacin lipopeptides - the effect of lengthening fatty acid chain.

In recent years, lipopeptides have received attention for their enhanced antimicrobial activity, especially against multi-drug resistant (MDR) pathogens. We have previously reported that the bacterial soil extracted, novel cyclic lipopeptide, battacin, and its synthetic analogues have enhanced antimicrobial activity against various Gram negative, Gram positive and fungal pathogens. In particular, the modification of the hydrophobic fatty acid chain and molecular structure has improved its activity. We have used small angle X-ray scattering (SAXS) and circular dichroism (CD) to characterise the low resolution structure of battacin lipopeptides containing covalently bonded fatty acid chains and the one without it. In the absence of fatty acids or with short fatty acid chain, the peptides adopted an extended random coil structure that is best described barbell-like shape, while fatty acids that are sufficiently long induced an aggregation into a ∼4.0 nm diameter core shell sphere. While the kinked structure found within this barbell shape may have a role in antimicrobial activities, the self-assembly of the battacin analogue with the longest fatty acid chain may have a correlation to the declined antibacterial activities.

Resolving solution conformations of the model semi-flexible polyelectrolyte homogalacturonan using molecular dynamics simulations and small-angle x-ray scattering.

The conformation of polyelectrolytes in the solution state has long been of interest in polymer science. Herein we utilize all atom molecular dynamics simulations (MD) and small-angle x-ray scattering experiments (SAXS) to elucidate the molecular structure of the model polyelectrolyte homogalacturonan. Several degrees of polymerization were studied and in addition partial methylesterification of the otherwise charge-carrying carboxyl groups was used in order to generate samples with varying intra-chain charge distributions. It is shown that at length scales above around 1nm the conformation of isolated chains has surprisingly little dependence on the charge distribution or the concentration of attendant monovalent salts, reflective of the intrinsic stiffness of the saccharide rings and the dynamical constraints of the glycosidic linkage. Indeed the conformation of isolated chains over all accessible length scales is well described by the atomic coordinates available from fibre diffraction studies. Furthermore, in more concentrated systems it is shown that, after careful analysis of the SAXS data, the form of the inter-particle effects heralded by the emergence of a so-called polyelectrolyte peak, can be extracted, and that this phenomena can be reproduced by multiple chain MD simulations.

Biomineralization of Calcium Phosphate and Calcium Carbonate within Iridescent Chitosan/Iota-Carrageenan Multilayered Films.

This work systematically explores the biomineralization of calcium phosphate (CaP) and carbonate (CaCO3) within chitosan/iota-carrageenan multilayer films. Multilayer films of chitosan and iota-carrageenan (up to 128-coupled layers) were prepared on glass substrates by a layer-by-layer dip-coating technique. Cryo-scanning electron microscopy revealed dense interfaces between the chitosan and iota-carrageenan layers with thicknesses in the range 250 and 350 nm in the hydrated state, accounting for the iridescent nature of multilayer films when wet. Immersion of the multilayered films in simulated body fluid or simulated seawater at 25 °C resulted in the mineralization of CaP and CaCO3, respectively, at the interfaces between the biopolymer layers and modified the iridescence of the films. Lamellar scattering features in small-angle neutron scattering measurements of the mineralized films provided evidence of the localized mineralization. Further evidence of this was found through the lack of change in the dynamic and static correlation lengths of the polymer networks within the bulk phase of the chitosan and iota-carrageenan layers. CaP mineralization occurred to a greater extent than CaCO3 mineralization within the films, evidenced by the higher lamellar density and greater rigidity of the CaP-mineralized films. Results provide valuable new insights into CaP and CaCO3 biomineralization in biopolymer networks.

Molecular Weight and Charge Density Effects of Guanidinylated Biodegradable Polycarbonates on Antimicrobial Activity and Selectivity.

Six guanidine functionalized aliphatic biodegradable polycarbonates with varying molecular weights and charge densities were synthesized via postsynthesis modification of alkyne containing polycarbonates using Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry. The concept of passive diluting group was to modify the cationic charge density of the polycarbonate without changing its hydrophilicity. Within the molecular weight range from 8000 to 30000 g mol-1, these guanidine polycarbonates exhibited broad-spectrum biocidal activity with low toxicity to red blood cells (RBCs). The lowest molecular weight homopolymer sample (PG-8k-100) showed the best antimicrobial activity (MIC = 40 µg/mL against Escherichia coli and MIC = 20 µg/mL against Staphylococcus epidermidis) and least RBC toxicity (0.6% hemolysis at MIC). Within the three guanidine charge densities from 20% to 70%, the low to medium dilution samples (PG-8k-7030 and PG-8k-5050) had no obvious loss in antimicrobial activities compared to the nondiluted control sample PG-8k-100. However, upon further dilution, PG-8k-2080 gave the lowest antimicrobial activity.

Chain shape and thin film behaviour of poly(thiophene)-graft-poly(acrylate urethane).

Electronic graft copolymers with conjugated polymer backbones are emerging as promising materials for various organic electronics. These materials combine the advantages of organic electronic materials, such as molecular tunability of opto-electronic and electrochemical properties, with solution processability and other 'designer' physical and mechanical properties imparted through the addition of grafted polymer side chains. Future development of such materials with complex molecular architecture requires a better understanding of the effect of molecular parameters, such as side chain length, on the structure and, in turn, on the electronic properties. In this study, poly(thiophene)-graft-poly(acrylate urethane) (PTh-g-PAU) was examined as a model system and we investigate the effect of side chain length on the overall shape and size in solution. Furthermore, the changes in the swelling behaviour of the graft copolymer thin films help in understanding their electrochemical redox properties.

Sugar-coated proteins: the importance of degree of polymerisation of oligo-galacturonic acid on protein binding and aggregation.

We have simplified the structural heterogeneity of protein-polysaccharide binding by investigating protein binding to oligosaccharides. The interactions between bovine beta-lactoglobulin A (ßLgA) and oligo-galacturonic acids (OGAs) with various numbers of sugar residues have been investigated with a range of biophysical techniques. We show that the ßLgA-OGA interaction is critically dependent on the length of the oligosaccharide. Isothermal titration calorimetry results suggest that a minimum length of 7 or 8 sugar residues is required in order to exhibit appreciable exothermic interactions with ßLgA - shorter oligosaccharides show no enthalpic interactions at any concentration ratio. When titrating ßLgA into OGAs with more than 7-8 sugar residues the sample solution also became turbid with increasing amounts of ßLgA, indicating the formation of macroscopic assemblies. Circular dichroism, thioflavin T fluorescence and small angle X-ray/neutron scattering experiments revealed two structural regimes during the titration. When OGAs were in excess, ßLgA formed discrete assemblies upon OGA binding, and no subsequent aggregation was observed. However, when ßLgA was present in excess, multi-scale structures were formed and this eventually led to the separation of the solution into two liquid-phases.

Apolipoprotein C-II Adopts Distinct Structures in Complex with Micellar and Submicellar Forms of the Amyloid-Inhibiting Lipid-Mimetic Dodecylphosphocholine.

The formation of amyloid deposits is a common feature of a broad range of diseases, including atherosclerosis, Alzheimer's disease, and Parkinson's disease. The basis and role of amyloid deposition in the pathogenesis of these diseases is still being defined, however an interesting feature of amyloidogenic proteins is that the majority of the pathologically associated proteins are involved in lipid homeostasis, be it in lipid transport, incorporation into membranes, or the regulation of lipid pathways. Thus, amyloid-forming proteins commonly bind lipids, and lipids are generally involved in the proper folding of these proteins. However, understanding of the basis for these lipid-related aspects of amyloidogenesis is lacking. Thus, we have used the apolipoprotein C-II amyloid model system in conjunction with x-ray and neutron scattering analyses to address this problem. Apolipoprotein C-II is a well-studied model system of systemic amyloid fibril formation, with a clear and well-defined pathway for fibril formation, where the effects of lipid interaction are characterized, particularly for the lipid mimetic dodecylphosphocholine. We show that the micellar state of an inhibitory lipid can have a very significant effect on protein conformation, with micelles stabilizing a particular α-helical structure, whereas submicellar lipids stabilize a very different dimeric, α-helical structure. These results indicate that lipids may have an important role in the development and progression of amyloid-related diseases.

Morphology of complexes formed between ß-lactoglobulin nanofibrils and pectins is influenced by the pH and structural characteristics of the pectins.

For the optimal use of ß-lactoglobulin nanofibrils as a raw material in biological composites an in-depth knowledge of their interactions with other constituents is necessary. To understand the effect of electrostatic interactions on the morphology of resulting complexes, ß-lactoglobulin nanofibrils were allowed to interact with pectins in which the amount of available negative charge was controlled by selecting their degree of methylesterification. In this study, citrus pectins having different degrees of methylesterification (∼48, 67, 86, and 97%) were selected and interacted with nanofibrils at pH 2 and pH 3, where they possess a net positive charge. Electrostatic complexes formed between ß-lactoglobulin nanofibrils and all pectin types, except for the sample having a degree of methylesterification of 97%. The morphology of these complexes, however, differed significantly with the degree of methylesterification of the pectin, its concentration, and the pH of the medium, revealing that distinct desired biological architectures can be attained relatively easily through manipulating the electrostatic interactions. Interestingly, the pectin with a degree of methylesterification of 86% was found to crosslink the ß-lactoglobulin nanofibrils into ordered 'nanotapes'.

Nonlinear Behavior of Gelatin Networks Reveals a Hierarchical Structure.

We investigate the strain hardening behavior of various gelatin networks-namely physical gelatin gel, chemically cross-linked gelatin gel, and a hybrid gel made of a combination of the former two-under large shear deformations using the pre-stress, strain ramp, and large amplitude oscillations shear protocols. Further, the internal structures of physical gelatin gels and chemically cross-linked gelatin gels were characterized by small angle neutron scattering (SANS) to enable their internal structures to be correlated with their nonlinear rheology. The Kratky plots of SANS data demonstrate the presence of small cross-linked aggregates within the chemically cross-linked network whereas, in the physical gelatin gels, a relatively homogeneous structure is observed. Through model fitting to the scattering data, we were able to obtain structural parameters, such as the correlation length (ξ), the cross-sectional polymer chain radius (R(c)) and the fractal dimension (d(f)) of the gel networks. The fractal dimension d(f) obtained from the SANS data of the physical and chemically cross-linked gels is 1.31 and 1.53, respectively. These values are in excellent agreement with the ones obtained from a generalized nonlinear elastic theory that has been used to fit the stress-strain curves. The chemical cross-linking that generates coils and aggregates hinders the free stretching of the triple helix bundles in the physical gels.

ß-Lactoglobulin nanofibrils can be assembled into nanotapes via site-specific interactions with pectin.

Controlling the self-assembly of individual supramolecular entities, such as amyloid fibrils, into hierarchical architectures enables the 'bottom-up' fabrication of useful bionanomaterials. Here, we present the hierarchical assembly of ß-lactoglobulin nanofibrils into the form of 'nanotapes' in the presence of a specific pectin with a high degree of methylesterification. The nanotapes produced were highly ordered, and had an average width of 180 nm at pH 3. Increasing the ionic strength or the pH of the medium led to the disassembly of nanotapes, indicating that electrostatic interactions stabilised the nanotape architecture. Small-angle X-ray scattering experiments conducted on the nanotapes showed that adequate space is available between adjacent nanofibrils to accommodate pectin molecules. To locate the interaction sites on the pectin molecule, it was subjected to endopolygalacturonase digestion, and the resulting products were analysed using capillary electrophoresis and size-exclusion chromatography for their charge and molecular weight, respectively. Results suggested that the functional pectin molecules carry short (<10 residues) enzyme-susceptible blocks of negatively charged, non-methylesterified galacturonic acid residues in the middle of their homogalacturonan backbones (and possibly near their ends), that specifically bind to sites on the nanofibrils. Blocking the interaction sites on the nanofibril surface using small oligomers of non-methylesterified galacturonic acid residues similar in size to the interaction sites of the pectin molecule decreased the nanotape formation, indicating that site-specific electrostatic interactions are vital for the cross-linking of nanofibrils. We propose a structural model for the pectin-cross-linked ß-lactoglobulin nanotapes, the elements of which will inform the future design of bionanomaterials.

Oxidative Damage to Biomimetic Membrane Systems: In Situ Fe(II)/Ascorbate Initiated Oxidation and Incorporation of Synthetic Oxidized Phospholipids.

Damage to cellular membranes from oxidative stress has been implicated in aging related diseases. We report the effects of oxidative damage on the structure and properties of biomimetic phospholipid membrane systems. Two oxidation methods were used, in situ oxidation initiated using Fe(II) and ascorbate, and the incorporation of a synthetic "oxidized" phospholipid, PoxnoPC, into biomimetic membranes. The biomimetic systems employed included multibilayer stacks, tethered bilayers, and phospholipid monolayers studied using a combination of reflectometry, attenuated total reflection infrared spectroscopy, electrochemical impedance spectroscopy, and neutron diffraction. We show that oxidation with Fe(II) and ascorbate caused an increase in the order of the membrane, attributed to cross-linking of the phospholipids, and a change in the electrical permeability of the membrane, but no significant impact on the thickness or completeness of the membrane. Incorporation of PoxnoPC, on the other hand, had a larger impact on the structure of the membrane. Inversion of the aldehyde-terminated truncated sn-2 chain of PoxnoPC into the head group region was observed, along with a slight decrease in the thickness and order of the membrane.

Structural mechanism of complex assemblies: characterisation of beta-lactoglobulin and pectin interactions.

Knowledge of how proteins and polysaccharides interact is the key to understanding encapsulation and emulsification in these composite systems and ultimately to understanding the structures of many biological network systems. As a model system we have studied ß-lactoglobulin A (ßLgA) interacting with pectins of various amounts and distribution patterns of charge. The studies were conducted at pH 4 at minimal ionic strength, where the ßLgA and the pectins are oppositely charged, resulting in an electrostatic attraction to each other. Isothermal titration calorimetry (ITC) experiments were performed to determine the thermodynamics associated with ßLgA-pectin titration. It was found that ßLgA only interacted with pectins with an adequate amount of charge, and that the complexation between ßLgA and pectin was a two-step process initially involving binding of the protein to available sites on the pectin, and subsequently binding of the protein onto the bound protein that has previously adsorbed. Circular dichroism (CD) and intrinsic tryptophan fluorescence were also measured of ßLgA during its interaction with the pectin samples, and show that the binding leads to significant conformational changes in ßLgA. An increase in the turbidity of the solution of the resultant complexes indicates the formation of large-scale interpolymer associations of the primary complexes mediated by protein-rich domains.