Bending, curling, and twisting in polymeric bilayers.

Polyolefin thermoplastic elastomer (POE) bilayers of varying length (L) to width (W) ratio are formed through traditional polymer processing. Each layer is completely isotropic but the bilayers have an elastic recovery mismatch such that when stretched, one layer recovers to a different extent than the other. Upon stretching bilayers from low to moderate strains and releasing the bilayer bends (curvature, κ, κ < 1/L). Stretching to moderate strain and releasing results in bilayer curling (1/L ≤ κ < 1/W). Finally, stretching to high strains and releasing such that κ ≥ 1/W results in twisting into a helix for L/W > 2π bilayers and rolling into a cylinder for L/W < 2π bilayers. Varying W can change the helical pitch, lp, of twisted bilayers. The twisted bilayer helical rise angle varies between θ = 60 and 90°. Metastability, i.e., bilayers that show a combination of the two behaviors, is observed at long absolute L or short absolute W. The bilayers are modeled using Euler-Bernoulli beam theory to show that the curvature can be predicted using the elastic recovery of the layer that recovers more.

Reducing the heterogeneity of xylan through processing.

Glycerol thermal processing (GTP) of hardwood biomass at temperatures between 200 and 240°C facilitated stepwise biopolymer fractionation, while limiting significant degradation of the major hemicellulose, glucuronoxylan, into water-extractable oligosaccharides. After GTP pretreatment and sequential water and organic solvent extraction, up to 80% of the initial xylan remained in the pretreated biomass. The majority of the xylan from GTP pretreated and water/solvent extracted biomass was removed using a mild alkali extraction and the composition was compared to xylan directly isolated from untreated hardwood. The precipitated xylan from the neutralized alkaline filtrate was isolated as a water insoluble xylan portion (WIX). The residual xylan dissolved in the neutralized filtrate was precipitated in cold methanol and recovered as the water soluble xylan portion (WSX). Results showed that xylan in WIX was in a polymeric form with a number average degree of polymerization (DP) over 100, whereas the WSX had a much lower average DP of 27 (ca) and contained more substitution. As the processing severity increased during GTP pretreatment, the proportion of WIX increased and the purity of the xylan within the WIX sample reached 84% based on compositional analysis. FT-IR analysis of WIX revealed that xylan isolated after GTP contained peaks related to a reduced carbonyl signal compared to the control. Furthermore, crude WSX contained less xylan with more lignin contamination at severe GTP conditions. The recovery of the xylan in two portions facilitated a preferential purification strategy resulting in WIX with an extremely narrow polydispersity index between 1.1 and 1.25, dependent upon the GTP severity. This study provided insight into fractionating higher molecular weight xylan that may serve value-added applications such as healthcare materials and advanced packaging.

Morphology selection via geometric frustration in chiral filament bundles.

In assemblies, the geometric frustration of a locally preferred packing motif leads to anomalous behaviours, from self-limiting growth to defects in the ground state. Here, we demonstrate that geometric frustration selects the equilibrium morphology of cohesive bundles of chiral filaments, an assembly motif critical to a broad range of biological and synthetic nanomaterials. Frustration of inter-filament spacing leads to optimal shapes of self-twisting bundles that break the symmetries of packing and of the underlying inter-filament forces, paralleling a morphological instability in spherical two-dimensional crystals. Equilibrium bundle morphology is controlled by a parameter that characterizes the relative costs of filament bending and the straining of cohesive bonds between filaments. This parameter delineates the boundaries between stable, isotropic cylindrical bundles and anisotropic, twisted-tape bundles. We also show how the mechanical and interaction properties of constituent amyloid fibrils may be extracted from the mesoscale dimensions of the anisotropic bundles that they form.

Enhanced enzymatic saccharification of pretreated biomass using glycerol thermal processing (GTP).

Biomass was heated (200-240°C) in the presence of glycerol, for 4-12 min, under shear to disrupt the native cell wall architecture. The impact of this method, named glycerol thermal processing (GTP), on saccharification efficiency of the hardwood Liquidambar styraciflua, and a control cellulose sample was studied as a function of treatment severity. Furthermore, the enzymatic conversion of samples with varying compositions was studied after extraction of the structural polymers. Interestingly, the sweet gum processed materials crystallinity index increased by 10% of the initial value. The experiments revealed that the residual lignin was not a barrier to limiting the digestibility of cellulose after pretreatment yielding up to 70% glucose based on the starting wood material. Further xylan removal greatly improved the cellulose hydrolysis rate, converting nearly 70% of the cellulose into glucose within 24h, and reaching 78% of ultimate glucan digestibility after 72 h.

A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers.

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.

The role of protein hydrophobicity in conformation change and self-assembly into large amyloid fibers.

It has been found that a short hydrophobic "template" peptide and a larger α-helical "adder" protein cooperatively self-assemble into micrometer sized amyloid fibers. Here, a common template of trypsin hydrolyzed gliadin is combined with six adder proteins (α-casein, α-lactalbumin, amylase, hemoglobin, insulin, and myoglobin) to determine what properties of the adder protein drive amyloid self-assembly. Utilizing Fourier Transform-Infrared (FT-IR) spectroscopy, the Amide I absorbance reveals that the observed decrease in α-helix with time is approximately equal to the increase in high strand density ß-sheet, which is indicative of amyloid formation. The results show that the hydrophobic moment is a good predictor of conformation change but the fraction of aliphatic amino acids within the α-helices is a better predictor. Upon drying, the protein mixtures form large amyloid fibers. The fiber twist is dependent on the aliphatic index and molecular weight of the adder protein. Here we demonstrate that it is possible to predict the propensity of an adder protein to unfold into an amyloid structure and to predict the fiber morphology, both from adder protein molecular features, which can be applied to the pragmatic engineering of large amyloid fibers.

Characterization of large amyloid fibers and tapes with Fourier transform infrared (FT-IR) and Raman spectroscopy.

Amyloids are self-assembled protein structures implicated in a host of neurodegenerative diseases. Organisms can also produce "functional amyloids" to perpetuate life, and these materials serve as models for robust biomaterials. Amyloids are typically studied using fluorescent dyes, Fourier transform infrared (FT-IR), or Raman spectroscopy analysis of the protein amide I region, and X-ray diffraction (XRD) because the self-assembled ß-sheet secondary structure of the amyloid can be easily identified with these techniques. Here, FT-IR and Raman spectroscopy analyses are described to characterize amyloid structures beyond just identification of the ß-sheet structure. It has been shown that peptide mixtures can self-assemble into nanometer-sized amyloid structures that then continue to self-assemble to the micrometer scale. The resulting structures are flat tapes of low rigidity or cylinders of high rigidity depending on the peptides in the mixture. By monitoring the aggregation of peptides in solution using FT-IR spectroscopy, it is possible to identify specific amino acids implicated in ß-sheet formation and higher order self-assembly. It is also possible to predict the final tape or cylinder morphology and gain insight into the structure's physical properties based on observed intermolecular interactions during the self-assembly process. Tapes and cylinders are shown to both have a similar core self-assembled ß-sheet structure. Soft tapes also have weak hydrophobic interactions between alanine, isoleucine, leucine, and valine that facilitate self-assembly. Rigid cylinders have similar hydrophobic interactions that facilitate self-assembly and also have extensive hydrogen bonding between glutamines. Raman spectroscopy performed on the dried tapes and fibers shows the persistence of these interactions. The spectroscopic analyses described could be generalized to other self-assembling amyloid systems to explain property and morphological differences.

Evolution of the amyloid fiber over multiple length scales.

The amyloid is a natural self-assembled peptide material comparable in specific stiffness to spider silk and steel. Throughout the literature there are many studies of the nanometer-sized amyloid fibril; however, peptide mixtures are capable of self-assembling beyond the nanometer scale into micrometer-sized fibers. Here, atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to observe the self-assembly of the peptide mixtures in solution for 20 days and the fibers upon drying. Beyond the nanometer scale, self-assembling fibers differentiate into two morphologies, cylindrical or rectangular cross-section, depending on peptide properties. Microscopic observations delineate a four stage self-assembly mechanism: (1) protofibril (2-4 nm high and 15-30 nm wide) formation; (2) protofibril aggregation into fibrils 6-10 nm high and 60-120 nm wide; (3) fibril aggregation into large fibrils and morphological differentiation where large fibrils begin to resemble the final fiber morphology of cylinders (WG peptides) or tapes (Gd:My peptides). WG large fibrils are 50 nm high and 480 nm wide and Gd:My large fibrils are 10 nm high and 150 nm wide; (4) micrometer-sized fiber formation upon drying at 480 h resulting in 18.0 µm diameter cylindrical fibers (WG peptides) and 14.0 µm wide and 6.0 µm thick flat tapes (Gd:My peptides). Evolution of the large fiber morphology can be rationalized on the basis of the peptide properties.

Peptide mixtures can self-assemble into large amyloid fibers of varying size and morphology.

Peptide mixtures spontaneously formed micrometer-sized fibers and ribbons from aqueous solution. Hydrolyzed gliadin produced short, slightly elliptical fibers while hydrolyzed wheat gluten, a mixture of gliadin and glutenin, formed round fibers of similar size. Mixing hydrolyzed gliadin with increasing molar amounts of myoglobin or amylase resulted in longer, wider fibers that transitioned from round to rectangular cross section. Fiber size, morphology, and modulus were controlled by peptide mixture composition. Fourier transform infrared (FT-IR) spectroscopy results showed that peptides experienced α to ß transitions forming an elementary cross-ß peptide secondary structure, indicative of amyloids. Large fiber formation was observed to be dependent on hydrophobic packing between constituent peptides. A model was developed to show how the fiber morphology was influenced by the peptides in the mixture.

Protein substitution affects glass transition temperature and thermal stability.

When proteins are removed from their native state they suffer from two deficiencies: (1) glassy behavior with glass transition temperatures (Tg) well above room temperature and (2) thermal instability. The glassy behavior originates in multiple hydrogen bonds between amino acids on adjacent protein molecules. Proteins, like most biopolymers, are thermally unstable. Substituting ovalbumin with linear and cyclic substituents using a facile nucleophilic addition reaction can affect Tg and thermal stability. More hydrophobic linear substituents lowered Tg by interrupting intermolecular interactions and increasing free volume. More hydrophilic and cyclic substituents increased thermal stability by increasing intermolecular interactions. In some cases, substituents instituted cross-linking between protein chains that enhanced thermal stability. Internal plasticization using covalent substitution and external plasticization using low molecular weight polar liquids show the same protein structural changes and a signature of plasticization is identified.

Conformational changes and molecular mobility in plasticized proteins.

Most biopolymers exist in a plasticized state, whether it is naturally with water or unnaturally with glycerol or other suitable polyol, to make a flexible material. We have found that the extent to which a biopolymer can be plasticized is dependent on its molecular and higher order structures outside of simply molecular weight. Lactalbumin, ovalbumin, corn zein, wheat gluten, and feather keratin were plasticized with glycerol from very low to very high amounts. The conformation of the proteins was monitored with Fourier transform-infrared (FT-IR) spectroscopy and X-ray powder diffraction (XRD) and correlated with the tensile modulus. Protein conformational changes were pronounced for polar proteins with a low amount of cysteine. FT-IR showed that the conformational changes resulted in ordering of the protein at low to moderate plasticization levels. For proteins with little resistance to conformational changes, additional small-scale ordering occurred around the glass transition, as observed in XRD. Accurate comparison of plasticized proteins was dependent on knowing whether or not the protein was glassy or rubbery at room temperature as no differences arose in the glassy state. The transition from glassy to rubbery behavior with plasticization level can be found from modulus, FT-IR, and XRD data.

Blends of cysteine-containing proteins.

Proteins such as keratin, lactalbumin, and gluten can be obtained from agricultural sources. These proteins contain the amino acid cysteine. Cysteine allows for the formation of inter- and intramolecular sulfur-sulfur bonds. It was found that cysteine-containing proteins have varied properties and can be blended together to form materials with the attributes of each polymer. The addition of wheat gluten to other proteins increases the strain to break or "toughness". The addition of lactalbumin increases the modulus and strength of blends. Birefringence shows that lactalbumin contains an added "structure" not found in the other proteins. Permeability studies reveal that one protein may dominate the transport of small molecules through the blend. Scanning electron microscopy shows that blends contain features of each protein and correlate with observed tensile properties.

Stearic acid solubility and cubic phase volume.

Stearic acid (SA) is highly soluble in structurally diverse solvents. SA/solvent packing within a (24.8 A)3 cubic volume explains the stoichiometry of SA solubility at multiple temperatures in multiple solvents. In the absence of solvent, the cubic volume contains 25 molecules at van der Waals distances from each other. At 55 degrees C, SA occupied half the cubic volume in saturated solution of four structurally diverse solvents. Below 4% SA/volume (e.g. in acetonitrile), the head and foot of each SA molecules on average is more than one solvent molecule away from the head and foot of a neighboring SA molecule. At 50% SA/cubic volume, -CH2- groups on SA molecules are separated from neighboring -CH2- groups on SA molecules by a monolayer of solvent molecules. Lowering the temperature from 55 to 25 degrees C, the volume fraction of SA decreased by a factor of 2 (or more) for every 6 degrees C. Lowering temperature increased the relative number of column of solvent molecules in the cubic phase, and correspondingly, the distance between SA molecules within the cubic volume increased. In three of five solvents, molecular mechanics calculations demonstrated the van der Waals stabilization that occurs from SA/SA affinity in the absence of solvent is similar in magnitude to the van der Waals stabilization from SA/solvent affinity. Methyl-t-butyl ether was less stabilized than hexane, acetone or methanol because the more bulky molecules packed less efficiently within the cubic volume. The most efficient/most stable packing however was still as columns of solvent between columns of SA. The efficiency and stability of SA and solvent packing optimal within the (24.8 A)3 cubic volume. Between 100 and 8% SA, multiple SA molecules present within the cubic volume function as SA aggregates. Both inter- and intra-cubic (phase) volume properties of SA aggregates coexist. Although acetonitrile and SA at the molecular level are both rod shaped, acetonitrile disrupted the packing of SA molecules within the cubic phase. The disrupted packing explains the much lower solubility of SA in acetonitrile than in the other solvents. The same molecular structures (e.g. methanol) can either stabilize or disrupt the packing of aggregated SA molecules, depending upon temperature. The mechanisms of aggregation within cubic volumes could also occur with structurally more complicated lipids. Aggregation and dispersion from such cubic phases could also be present in more complex chemical and/or macromolecular environments.

Effect of formic acid exposure on keratin fiber derived from poultry feather biomass.

Converting poultry feather biomass into useful products presents a new avenue of utilization of agricultural waste material. However, not much is understood about the poultry feather structure or methods to process it. In this study, formic acid vapor is systematically allowed to penetrate the feather fiber structure, which is composed of keratin. The diffusion kinetics show Fickian behavior during absorption. After very long times, i.e., greater than 10(3)h, the absorption experiments are stopped and the formic acid is allowed to desorb from the keratin material. The desorption kinetics of formic acid out of the keratin fiber do not mirror the absorption kinetics, indicating a change in the keratin microstructure. DSC and NMR spectroscopy analyses on the keratin fiber show a reduction in the area of the crystalline melting peak and solubilization of amino acids upon formic acid exposure. This indicates that the crystallinity is disrupted resulting in more amorphous fraction in the keratin polymer.