Effect of pH and protein composition on proteolysis of goat milk proteins by pepsin and pancreatin.

The roles of protein composition, pH and enzymes in goat milk protein hydrolysis is still unclear and the proteolysis of low abundant goat milk proteins has received limited attention. The aim of this study was to study the impact of protein composition and proteolytic conditions on goat milk protein hydrolysis in a simplified digestion model. Both whole milk and infant formula were hydrolyzed at pH 2 and 4, using pepsin as well as pepsin combined with pancreatin. Intact proteins were separated from digests using spin filters, followed by bottom-up proteomics of the separated proteins. Results show that under all conditions, caseins are hydrolyzed quickly. Goat casein hydrolysis in infant formula was slightly faster than in goat whole milk, possibly due to less casein coagulation during pepsin hydrolysis at both pH 2 and 4. Several low abundant immunoactive goat milk proteins, especially immunoglobulins, GLYCAM-1 and osteopontin, resisted proteolysis more than high abundant proteins, independent of the pH and enzyme used for hydrolysis. Fast hydrolysis of casein and slow hydrolysis of immunoactive proteins may indicate a good balance between protein utilization and protection of the infant by goat milk proteins.

Gastric protein digestion of goat and cow milk infant formula and human milk under simulated infant conditions.

The protein digestion kinetics of goat milk infant formula (GMF) is previously shown to be more comparable to that of human milk (HM) than cow milk infant formula (CMF). To evaluate whether gastric behaviour contributes to differences in protein digestion kinetics, fresh HM, a GMF and a CMF were subjected to in vitro gastric digestion simulating infant conditions. Coagulation behaviour, particle size distribution and viscosity of the digesta were evaluated. After centrifugation of the digesta, total solids and protein distribution, and protein hydrolysis in the cream, serum and pellet fraction were investigated. The GMF and CMF were in general similar with respect to physicochemical and protein breakdown properties. However, a number of notable differences in physicochemical behaviour were observed, which may contribute to faster initial protein digestion of GMF. HM behaved differently from both formulas. These differences provide new insights into the possibilities for improvement of infant formulas.

Mechanics of composite hydrogels approaching phase separation.

For polymer-particle composites, limited thermodynamic compatibility of polymers and particles often leads to poor dispersal and agglomeration of the particles in the matrix, which negatively impacts the mechanics of composites. To study the impact of particle compatibility in polymer matrices on the mechanical properties of composites, we here study composite silica- protein based hydrogels. The polymer used is a previously studied telechelic protein-based polymer with end groups that form triple helices, and the particles are silica nanoparticles that only weakly associate with the polymer matrix. At 1mM protein polymer, up to 7% of silica nanoparticles can be embedded in the hydrogel. At higher concentrations the system phase separates. Oscillatory rheology shows that at high frequencies the particles strengthen the gels by acting as short-lived multivalent cross-links, while at low frequencies, the particles reduce the gel strength, presumably by sequestering part of the protein polymers in such a way that they can no longer contribute to the network strength. As is generally observed for polymer/particle composites, shear-induced polymer desorption from the particles leads to a viscous dissipation that strongly increases with increasing particle concentration. While linear rheological properties as function of particle concentration provide no signals for an approaching phase separation, this is very different for the non-linear rheology, especially fracture. Strain-at-break decreases rapidly with increasing particle concentration and vanishes as the phase boundary is approached, suggesting that the interfaces between regions of high and low particle densities in composites close to phase separation provide easy fracture planes.

Recombinant Protein Polymers for Colloidal Stabilization and Improvement of Cellular Uptake of Diamond Nanosensors.

Fluorescent nanodiamonds are gaining increasing attention as fluorescent labels in biology in view of the fact that they are essentially nontoxic, do not bleach, and can be used as nanoscale sensors for various physical and chemical properties. To fully realize the nanosensing potential of nanodiamonds in biological applications, two problems need to be addressed: their limited colloidal stability, especially in the presence of salts, and their limited ability to be taken up by cells. We show that the physical adsorption of a suitably designed recombinant polypeptide can address both the colloidal stability problem and the problem of the limited uptake of nanodiamonds by cells in a very straightforward way, while preserving both their spectroscopic properties and their excellent biocompatibility.

Enhanced stiffness of silk-like fibers by loop formation in the corona leads to stronger gels.

We study the self-assembly of protein polymers consisting of a silk-like block flanked by two hydrophilic blocks, with a cysteine residue attached to the C-terminal end. The silk blocks self-assemble to form fibers while the hydrophilic blocks form a stabilizing corona. Entanglement of the fibers leads to the formation of hydrogels. Under oxidizing conditions the cysteine residues form disulfide bridges, effectively connecting two corona chains at their ends to form a loop. We find that this leads to a significant increase in the elastic modulus of the gels. Using atomic force microscopy, we show that this stiffening is due to an increase of the persistence length of the fibers. Self-consistent-field calculations indicate a slight decrease of the lateral pressure in the corona upon loop formation. We argue that this small decrease in the repulsive interactions affects the stacking of the silk-like blocks in the core, resulting in a more rigid fiber.

Nanoparticle-Templated Formation and Growth Mechanism of Curved Protein Polymer Fibrils.

We investigated the growth of biosynthetic protein polymers with templated curvature on pluronic nanospheres. The protein has a central silk-like block containing glutamic residues (S(E)) and collagen-like end-blocks (C). The S(E) blocks stack into filaments when their charge is removed (pH <5). Indeed, at low pH curved and circular fibers are formed at the surface of the nanospheres, which keep their shape after removal of the pluronics. The data reveal the mechanism of the templated fibril-growth: The growth of protein assemblies is nucleated in solution; small protein fibrils adsorb on the nanospheres, presumably due to hydrogen bond formation between the silk-like blocks and the pluronic PEO blocks. The surface of the pluronic particles templates further growth. At relatively low protein/pluronic weight ratios, only a fraction of the nanospheres bears protein fibers, pointing to a limiting amount of nuclei in solution. Because the nanospheres capture fibrils at an early stage of growth, they can be used to separate growth and nucleation rates in protein fibril formation. Moreover, the nanoparticle-templated growth of stable curved fibers opens ways to build proteinaceous nanocapsules from designed protein polymers.

Reversible Temperature-Switching of Hydrogel Stiffness of Coassembled, Silk-Collagen-Like Hydrogels.

Recombinant protein polymers, which can combine different bioinspired self-assembly motifs in a well-defined block sequence, have large potential as building blocks for making complex, hierarchically structured materials. In this paper we demonstrate the stepwise formation of thermosensitive hydrogels by combination of two distinct, orthogonal self-assembly mechanisms. In the first step, fibers are coassembled from two recombinant protein polymers: (a) a symmetric silk-like block copolymer consisting of a central silk-like block flanked by two soluble random coil blocks and (b) an asymmetric silk-collagen-like block copolymer consisting of a central random-coil block flanked on one side by a silk-like block and on the other side a collagen-like block. In the second step, induced by cooling, the collagen-like blocks form triple helices and thereby cross-link the fibers, leading to hydrogels with a thermo-reversibly switchable stiffness. Our work demonstrates how complex self-assembled materials can be formed through careful control of the self-assembly pathway.

Equivalent pathways in melting and gelation of well-defined biopolymer networks.

We use multiple particle tracking microrheology to study the melting and gelation behavior of well-defined collagen-inspired designer biopolymers expressed by the transgenic yeast P. Pastoris. The system consists of a hydrophilic random coil-like middle block and collagen-like end block. Upon cooling, the end blocks assemble into well-defined transient nodes with exclusively 3-fold functionality. We apply the method of time-cure superposition of the mean-square displacement of tracer beads embedded in the biopolymer matrix to study the kinetics and thermodynamics of approaching the gel point from both the liquid and the solid side. The melting point, gel point, and critical relaxation exponents are determined from the shift factors of the mean-square displacement and we discuss the use of dynamic scaling exponents to correctly determine the critical transition. Critical relaxation exponents obtained for different concentrations in both systems are compared with the currently existing dynamic models in literature. In our study, we find that, while the time scales of gelation and melting are different by orders of magnitude, and show inverse dependence on concentration, that the pathways followed are completely equivalent.

Synergistic stiffening in double-fiber networks.

Many biological materials are composite structures, interpenetrating networks of different types of fibers. The composite nature of such networks leads to superior mechanical properties, but the origin of this mechanical synergism is still poorly understood. Here we study soft composite networks, made by mixing two self-assembling fiber-forming components. We find that the elastic moduli of the composite networks significantly exceed the sum of the moduli of the two individual networks. This mechanical enhancement is in agreement with recent simulations, where it was attributed to a suppression of non-affine deformation modes in the most rigid fiber network due to the reaction forces in the softer network. The increase in affinity also causes a loss of strain hardening and an increase in the critical stress and stain at which the network fails.