Renko de Vries

Design and self-assembly of simple coat proteins for artificial viruses

Viruses are among the simplest biological systems and are highly effective vehicles for the delivery of genetic material into susceptible host cells. Artificial viruses can be used as model systems for providing insights into natural viruses and can be considered a testing ground for developing artificial life. Moreover, they are used in biomedical and biotechnological applications, such as targeted delivery of nucleic acids for gene therapy and as scaffolds in material science. In a natural setting, survival of viruses requires that a significant fraction of the replicated genomes be completely protected by coat proteins. Complete protection of the genome is ensured by a highly cooperative supramolecular process between the coat proteins and the nucleic acids, which is based on reversible, weak and allosteric interactions only. However, incorporating this type of supramolecular cooperativity into artificial viruses remains challenging. Here, we report a rational design for a self-assembling minimal viral coat protein based on simple polypeptide domains. Our coat protein features precise control over the cooperativity of its self-assembly with single DNA molecules to finally form rod-shaped virus-like particles. We confirm the validity of our design principles by showing that the kinetics of self-assembly of our virus-like particles follows a previous model developed for tobacco mosaic virus. We show that our virus-like particles protect DNA against enzymatic degradation and transfect cells with considerable efficiency, making them promising delivery vehicles.

The influence of ligand valency on aggregation mechanisms for inhibiting bacterial toxins.

Divalent and tetravalent analogues of ganglioside GM1 are potent inhibitors of cholera toxin and Escherichia coli heat‐labile toxin. However, they show little increase in inherent affinity when compared to the corresponding monovalent carbohydrate ligand. Analytical ultracentrifugation and dynamic light scattering have been used to demonstrate that the multivalent inhibitors induce protein aggregation and the formation of space‐filling networks. This aggregation process appears to arise when using ligands that do not match the valency of the protein receptor. While it is generally accepted that multivalency is an effective strategy for increasing the activity of inhibitors, here we show that the valency of the inhibitor also has a dramatic effect on the kinetics of aggregation and the stability of intermediate protein complexes. Structural studies employing atomic force microscopy have revealed that a divalent inhibitor induces head‐to‐head dimerization of the protein toxin en route to higher aggregates.

Preparation and characterization of oxidized starch polymer microgels for encapsulation and controlled release of functional ingredients.

A novel biocompatible and biodegradable microgel system has been developed for controlled uptake and release of especially proteins. It contains TEMPO-oxidized potato starch polymers, which are chemically cross-linked by sodium trimetaphosphate (STMP). Physical chemical properties have been determined for microgels of different weight ratios of cross-linker to polymer (0.10, 0.15, 0.20, 0.30, and 0.40) and degrees of oxidation (30, 50, 70, and 100%). The charge density of the microgels as determined by proton titration is found to be in good agreement with the expected degree of oxidation (DO). The electrophoretic mobility of the microgel particles is used as a qualitative indicator of the pore size and scales with microgel swelling capacity as expected. The swelling capacity increases with increasing pH and decreasing salt concentration. Preliminary data for the uptake of the globular protein lysozyme by the microgels show it increases with increasing DO and decreasing cross-linker to polymer ratio. Highly charged microgels with intermediate cross-linker to polymer ratios (0.15 and 0.2) are found to be optimal for encapsulating lysozyme.

Salt-induced release of lipase from polyelectrolyte complex micelles

With the aim to gain insight into the possible applicability of protein-filled polyelectrolyte complex micelles under physiological salt conditions, we studied the behavior of these micelles as a function of salt concentration. The micelles form by electrostatically driven co-assembly from strong cationic block copolymers poly(2-methyl vinyl pyridinium)41-block- poly(ethylene oxide)205, weak anionic homopolymers poly(acrylic acid)139, and negatively charged lipase molecules. The formation and disintegration of these micelles were studied with dynamic light scattering (DLS), by means of composition and salt titrations, respectively. The latter measurements revealed differences between disintegration of lipase-filled and normal polyelectrolyte complex micelles. These data, together with small angle neutron scattering (SANS) measurements provide indications that lipase is gradually released with increasing salt concentration. From the SANS data a linear relation between the intensity at q = 0 and the volume of the cores of the micelles at different salt concentrations was derived, indicating a loss of volume of the micelles due to the release of lipase molecules. It was estimated that beyond 0.12 M NaCl all lipase molecules are released.

Flexible Polymer-Induced Condensation and Bundle Formation of DNA and F-Actin Filaments

A simple semi-empirical theory is developed for the ionic strength dependence of the flexible polymer-induced condensation of semiflexible polyelectrolytes such as DNA and F-actin filaments. Critical concentrations of flexible polymer needed for condensation are calculated by comparing the free energies of inserting the semiflexible polyelectrolytes in a solution of flexible polymers, respectively, in their free state, and in their condensed state. Predictions of the theory are compared to experimental data on the condensation of DNA and F-actin filaments induced by the flexible polymer poly(ethylene oxide). The theory also predicts that reentrant decollapse is possible at low ionic strength and high concentrations of flexible polymer, as observed for DNA.

Lysozyme uptake by oxidized starch polymer microgels.

With the aim of determining suitable conditions for uptake and release of globular proteins on microgels, we studied the interaction between phosphated, highly cross-linked, negatively charged oxidized potato starch polymer (OPSP) microgel particles and lysozyme from hen eggs. Our microgel shows a typical protein-induced deswelling behavior for charged microgels. The protein distributes rather homogenously through the microgel. We found that at low salt concentration the saturation protein uptake Gammasat increases with increasing pH. This is because the binding capacity is mainly determined by charge compensation: with increasing pH, the (positive) charge on the lysozyme molecules decreases, while the (negative) charge of the microgel particles increases. Therefore, more protein molecules are needed to compensate for the charge on the gel and the binding capacity increases. The protein binding affinity, however, decreases sharply with increasing pH, presumably because this affinity is mainly sensitive to the lysozyme charge density. At high pH the binding affinity is relatively low, and by adding salt, the protein can easily be released from the gel. This leads to a maximum in the curves of Gammasat versus pH, and this maximum shifts to lower pH values with increasing ionic strength. We conclude that, for protein uptake and release applications, the present system works best around pH 5 due to a sufficiently high binding affinity and a sufficiently high binding capacity.

Salt-Induced Disintegration of Lysozyme-Containing Polyelectrolyte Complex Micelles

The salt-induced disintegration of lysozyme-filled polyelectrolyte complex micelles, consisting of positively charged homopolymers (PDMAEMA150), negatively charged diblock copolymers (PAA42-PAAm417), and lysozyme, has been studied with dynamic light scattering (DLS) and small-angle neutron scattering (SANS). These measurements show that, from 0 to 0.2 M NaCl, both the hydrodynamic radius (Rh) and the core radius (Rcore) decrease with increasing salt concentration. This suggests that the micellar structures rearrange. Moreover, from approximately 0.2 to 0.4 M NaCl the light-scattering intensity is constant. In this salt interval, the hydrodynamic radius increases, has a maximum at 0.3 M NaCl, and subsequently decreases. This behavior is observed in both a lysozyme-containing system and a system without lysozyme. The SANS measurements on the lysozyme-filled micelles do not show increased intensity or a larger core radius at 0.3 M NaCl. This indicates that from 0.2 to 0.4 M NaCl another structure is formed, consisting of just the diblock copolymer and the homopolymer, because at 0.12 M NaCl the lysozyme-PAA42-PAAm417 complex has disintegrated. One may expect that the driving force for the formation of the complex in this salt range is other than electrostatic.

Theoretical modeling of the kinetics of fibrilar aggregation of bovine β-lactoglobulin at pH 2

The authors propose a kinetic model for the heat-induced fibrilar aggregation of bovine beta-lactoglobulin at pH 2.0. The model involves a nucleation step and a simple addition reaction for the growth of the fibrils, as well as a side reaction leading to the irreversible denaturation and inactivation of a part of the protein molecules. For the early stages of the aggregation reaction, the authors obtain an analytical solution of the model. In agreement with their experimental results, the model predicts a critical protein concentration below where almost no fibrils are formed. The model agrees well with their experimental data from in situ light scattering. By fitting the experimental data with the model, the authors obtain the ionic strength dependent kinetic rate constants for beta-lactoglobulin fibrilar aggregation and the size of the critical nucleus.

Coating of single DNA molecules by genetically engineered protein diblock copolymers.

Coating DNA is an effective way to modulate its physical properties and interactions. Current chemosynthetic polymers form DNA aggregates with random size and shape. In this study, monodisperse protein diblock copolymers are produced at high yield in recombinant yeast. They carry a large hydrophilic colloidal block (≈400 amino acids) linked to a short binding block (≈12 basic amino acids). It is demonstrated that these protein polymers complex single DNA molecules as highly stable nanorods, reminiscent of cylindrical viruses. It is proposed that inter- and intramolecular bridging of DNA molecules are prevented completely by the small size of the binding block attached to the large colloidal stability block. These protein diblocks serve as a scaffold that can be tuned for application in DNA-based nanotechnology.

From micelles to fibers: balancing self-assembling and random coiling domains in pH-responsive silk-collagen-like protein-based polymers

We study the self-assembly of genetically engineered protein-based triblock copolymers consisting of a central pH-responsive silk-like middle block (SHn, where SH is a silk-like octapeptide, (GA)3GH and n is the number of repeats) flanked by hydrophilic random coil outer blocks (C2). Our previous work has already shown that triblocks with very long midblocks (n = 48) self-assemble into long, stiff protein filaments at pH values where the middle blocks are uncharged. Here we investigate the self-assembly behavior of the triblock copolymers for a range of midblock lengths, n = 8, 16, 24, 48. Upon charge neutralization of SHn by adjusting the pH, we find that C2SH8C2 and C2SH16C2 form spherical micelles, whereas both C2SH24C2 and C2SH48C2 form protein filaments with a characteristic beta-roll secondary structure of the silk midblocks. Hydrogels formed by C2SH48C2 are much stronger and form much faster than those formed by C2SH24C2. Enzymatic digestion of much of the hydrophilic outer blocks is used to show that with much of the hydrophilic outer blocks removed, all silk-midblocks are capable of self-assembling into stiff protein filaments. In that case, reduction of the steric repulsion by the hydrophilic outer blocks also leads to extensive fiber bundling. Our results highlight the opposing roles of the hydrophilic outer blocks and central silk-like midblocks in driving protein filament formation. They provide crucial information for future designs of triblock protein-based polymers that form stiff filaments with controlled bundling, that could mimick properties of collagen in the extracellular matrix.