Interplay between Folding and Assembly of Fibril-Forming Polypeptides.

Polypeptides can self-assemble into hierarchically organized fibrils consisting of a stack of individually folded polypeptides driven together by hydrophobic interaction. Using a coarse grained model, we systematically studied this self-assembly as a function of temperature and hydrophobicity of the residues on the outside of the building block. We find the self-assembly can occur via two different pathways - a random aggregation-folding route, and a templated-folding process - thus indicating a strong coupling between folding and assembly. The simulation results can explain experimental evidence that assembly through stacking of folded building blocks is rarely observed, at the experimental concentrations. The model thus provides a generic picture of hierarchical fibril formation.Comment: Accepted in Physical Review Letter

Self-consistent field theory of protein adsorption in a non-Gaussian polyelectrolyte brush

To describe adsorption of globular protein molecules in a polyelectrolyte brush we use the strong-stretching approximation of the Edwards self-consistent field equation, combined with corrections for a non-Gaussian brush. To describe chemical potentials in this mixture of (globular) species of widely varying sizes (ions, brush polyelectrolyte segments, globular protein molecules), we use the Boublik-Mansoori-Carnahan-Starling-Leland equation of state derived for polydisperse mixtures of spherical particles. The polyelectrolyte chain is described in this approach as a string of beads with the beads of a size related to the chain diameter. We use the one-dimensional Poisson equation to describe the electrostatic field and include the ionizable character of both the brush polyions and the protein molecules. This model explains the experimental observation of high amounts of protein adsorption in a polyacid brush for pH values above the isoelectric point of the protein as being due to charge reversal of the protein molecules upon entry in the brush. We find a distinct minimum in protein concentration near the edge of the brush. With increasing pH this barrier to protein transfer becomes larger, but much less so when we increase the ionic strength, a difference that might relate to an experimentally observed difference in the protein release rate in these two cases. A free energy analysis shows that the release of small ions from the brush and the increase of brush ionization are the two driving forces for protein adsorption in a like-charged brus

Process design for enzymatic peptide synthesis in near-anhydrous organic media

This work is a case study on a process design for enzymatic peptide synthesis, which is based on and inspired by previously established data about the Alcalase-catalyzed coupling of an amino acid amide and a chemically synthesized activated N-protected amino acid carbamoylmethyl ester in near-anhydrous tetrahydrofuran. The choices with regard to Alcalase formulation, the type of reactor, method of controlling the water content, and whether or not to recycle the enzyme, are discussed. In addition, an estimate is given for the reactor size, volumes of solvent, amount of substrate, enzyme and molecular sieves, needed in order to meet a specific demand for peptides. We believe that this case study gives a good indication of the various choices that have to be made when designing a process for enzymatic peptide synthesis and the implications of these choices

Phase behavior of mixtures of oppositely charged nanoparticles: Heterogeneous Poisson-Boltzmann cell model applied to lysozyme and succinylated lysozyme

We study the phase behavior of mixtures of oppositely charged nanoparticles, both theoretically and experimentally. As an experimental model system we consider mixtures of lysozyme and lysozyme that has been chemically modified in such a way that its charge is nearly equal in magnitude but opposite in sign to that of unmodified lysozyme. We observe reversible macroscopic phase separation that is sensitive not only to protein concentration and ionic strength, but also to temperature. We introduce a heterogeneous Poisson-Boltzmann cell model that generally applies to mixtures of oppositely charged nanoparticles. To account for the phase behavior of our experimental model system, in addition to steric and electrostatic interactions, we need to include a temperature-dependent short-ranged interaction between the lysozyme molecules, the exact origin of which is unknown. The strength and temperature dependence of the short-ranged attraction is found to be of the same order of magnitude as that between unmodified lysozyme molecules. The presence of a rather strong short-ranged attraction in our model system precludes the formation of colloidal liquid phases (or complex coacervates) such as those typically found in mixtures of globular protein molecules and oppositely charged polyelectrolytes

Use of polysaccharides to control protein adsorption to the air-water interface

In order to understand foaming behaviour of mixed protein/anionic polysaccharide solutions, we investigated the effect of beta-lactoglobulin/pectin interaction in the bulk on beta-lactoglobulin adsorption to the air-water interface. Adsorption kinetics were evaluated by following surface pressure development in time of several pure protein solutions and of mixed protein/polysaccharide solutions using an automated drop tensiometer (ADT). It was found that complexation of proteins with polysaccharides can slow down the kinetics of surface pressure development by at least a factor 100; and greatly diminish foam formation. In contrast, a five times acceleration in the increase of surface pressure was observed in other cases. We propose a mechanism for protein adsorption from mixed protein/polysaccharide solutions. Effects of ionic strength, pH and mixing ratio on this mechanism were studied for mixtures of beta-lactoglobulin and low methoxyl pectin, whereas other proteins and anionic polysaccharides were used to explore the role of protein and polysaccharide charge density and distribution. Whereas the possibilities to change system parameters like ionic strength or pH are limited in food related systems, selecting a suitable combination of protein and polysaccharide offers a broad opportunity to control protein adsorption kinetics and with that foam formation

Surface charge regulation upon polyelectrolyte adsorption, hematite, polystyrene sulfonate, surface charge regulation - Theoretical calculations and hematite-poly(styrene sulfonate) system

The charge regulation of a mineral surface upon adsorption of a strong polyelectrolyte is studied theoretically and experimentally. Self-consistent-field calculations were done to evaluate the charge characteristics of a model oxide surface in the absence and presence of a linear strong polyelectrolyte. The calculations show high affinity adsorption isotherms for the polyelectrolyte at different pH. At the adsorption plateau the surface charge is overcompensated by the charge of the adsorbed polyelectrolyte. The increase in surface charge upon polyelectrolyte adsorption is substantial. For a bare surface a similar adjustment would require a pH change by about three units. At a given pH and salt concentration the proton co-adsorption ratio increases with the adsorbed polyelectrolyte charge till the charge compensation point is reached and then it decreases again. The measured adsorption isotherms of linear poly(styrene sulfonate) (PSS) on hematite do not show a high affinity character. This might be due to fast flocculation and a non-equilibrium polymer conformation at the surface. At pH 7 the adsorption plateau is reached and the surface charge is overcompensated by the adsorbed polyelectrolyte charge. The adsorption of PSS increases the surface charge, similarly as in the calculations. The increase of the surface charge with increasing PSS adsorption is about linear up to the charge compensation point and decreases beyond the charge compensation poin

Shape-Memory Effects in Biopolymer Networks with Collagen-Like Transient Nodes

In this article we study shape-memory behavior of hydrogels, formed by biodegradable and biocompatible recombinant telechelic polypeptides, with collagen-like end blocks and a random coil-like middle block. The programmed shape of these hydrogels was achieved by chemical cross-linking of lysine residues present in the random coil. This led to soft networks, which can be stretched up to 200% and “pinned” in a temporary shape by lowering the temperature and allowing the collagen-like end blocks to assemble into physical nodes. The deformed shape of the hydrogel can be maintained, at room temperature, for several days, or relaxed within a few minutes upon heating to 50 °C or higher. The presented hydrogels could return to their programmed shape even after several thermomechanical cycles, indicating that they remember the programmed shape. The kinetics of shape recovery at different temperatures was studied in more detail and analyzed using a mechanical model composed of two springs and a dashpot

Shape-Memory Effects in Biopolymer Networks with Collagen-Like Transient Nodes

In this article we study shape-memory behavior of hydrogels, formed by biodegradable and biocompatible recombinant telechelic polypeptides, with collagen-like end blocks and a random coil-like middle block. The programmed shape of these hydrogels was achieved by chemical cross-linking of lysine residues present in the random coil. This led to soft networks, which can be stretched up to 200% and “pinned” in a temporary shape by lowering the temperature and allowing the collagen-like end blocks to assemble into physical nodes. The deformed shape of the hydrogel can be maintained, at room temperature, for several days, or relaxed within a few minutes upon heating to 50 °C or higher. The presented hydrogels could return to their programmed shape even after several thermomechanical cycles, indicating that they remember the programmed shape. The kinetics of shape recovery at different temperatures was studied in more detail and analyzed using a mechanical model composed of two springs and a dashpot