Dominique M. E. Thies-Weesie

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.

Uniaxial and biaxial liquid crystal phases in colloidal dispersions of board-like particles

Dispersions of board-like goethite (α-FeOOH) particles with short-range repulsive interaction form a versatile colloidal model system, showing a nematic, smectic A and columnar phase. In high magnetic fields a biaxial nematic phase is induced with the shortest dimension of the particles aligned along the field. Moreover, if particles have a shape almost exactly in between rod-like and plate-like they can spontaneously, without external magnetic field, form biaxial nematic and biaxial smectic A phases, which is in accordance with theoretical predictions. The macroscopic domains were oriented by a magnetic field and their structure was revealed by small-angle X-ray scattering. Our results suggest that biaxial phases can be readily obtained by a proper choice of the particle shape.

Structural transitions of hard-sphere colloids studied by spin-echo small-angle neutron scattering

The structure of hard-sphere colloidal suspensions is measured at different concentrations using the recently developed spin-echo small-angle neutron scattering (SESANS) technique. It is shown that SESANS measures real-space correlations ranging from the size of a single particle for a dilute suspension to several particle diameters for a concentrated suspension, glass and crystalline state.

Influence of polydispersity on the phase behavior of colloidal goethite

The effect of fractionation on the phase behavior of colloidal goethite dispersions with different polydispersities (17%, 35%, and 55% in length) has been studied by small angle x-ray scattering and transmission electron microscopy. All systems show at least nematic and smectic phases. The occurrence of the latter phase at such a high polydispersity is remarkable. It is shown that in the highly polydisperse systems strong fractionation occurs, which is able to reduce the local length polydispersity up to a factor of 2. A columnar phase was only found in the 35% and 55% polydisperse systems. It seems that the columnar phase accommodates the particles that do not fit into the smectic layers and, thus, reduces the length polydispersity within the smectic phase even further. The fact that a columnar phase was not found in the system of lowest polydispersity indicates that the smectic phase is the most stable phase at higher concentrations.

Structural Insight into Multivalent Galactoside Binding to Pseudomonas aeruginosa Lectin LecA

Multivalent galactosides inhibiting Pseudomonas aeruginosa biofilms may help control this problematic pathogen. To understand the binding mode of tetravalent glycopeptide dendrimer GalAG2 [(Gal-β-OC6H4CO-Lys-Pro-Leu)4(Lys-Phe-Lys-Ile)2Lys-His-Ile-NH2] to its target lectin LecA, crystal structures of LecA complexes with divalent analog GalAG1 [(Gal-β-OC6H4CO-Lys-Pro-Leu)2Lys-Phe-Lys-Ile-NH2] and related glucose-triazole linked bis-galactosides 3u3 [Gal-β-O(CH2)n-(C2HN3)-4-Glc-β-(C2HN3)-[β-Glc-4-(N3HC2)]2-(CH2)n-O-β-Gal (n = 1)] and 5u3 (n = 3) were obtained, revealing a chelate bound 3u3, cross-linked 5u3, and monovalently bound GalAG1. Nevertheless, a chelate bound model better explaining their strong LecA binding and the absence of lectin aggregation was obtained by modeling for all three ligands. A model of the chelate bound GalAG2·LecA complex was also obtained rationalizing its unusually tight LecA binding (KD = 2.5 nM) and aggregation by lectin cross-linking. The very weak biofilm inhibition with divalent LecA inhibitors suggests that lectin aggregation is necessary for biofilm inhibition by GalAG2, pointing to multivalent glycoclusters as a unique opportunity to control P. aeruginosa biofilms.

Sedimentation equilibria of ferrofluids: I. Analytical centrifugation in ultrathin glass capillaries

Analytical centrifugation is used for the first time to measure sedimentation equilibrium concentration profiles of a ferrofluid, a concentrated colloidal dispersion of strongly absorbing magnetic nanoparticles. To keep the optical absorbance from becoming too strong, the optical path length is restricted to 50 μm by placing the dispersion in a flat glass capillary. The concentration profile is kept from becoming too steep, despite the relatively high buoyant mass of the nanoparticles, by making novel use of a low-velocity analytical centrifuge that was not designed to measure equilibrium profiles. The experimental approach is validated by comparison with profiles obtained using an analytical ultracentrifuge. At concentrations of a few hundred grams per liter, the osmotic pressures calculated from the equilibrium profiles are lower than expected for hard spheres or non-interacting particles, due to magnetic dipolar interactions. By following the presented experimental approach, it will now also be possible to characterize the interparticle interactions of other strongly absorbing colloidal particles not studied before by analytical centrifugation.

Simple Rectangular Columnar Phase of Goethite Nanorods and Its Martensitic Transition to the Centered Rectangular Columnar Phase

Using high-resolution small-angle X-ray scattering, we observed a new type of the columnar phase with a simple rectangular (R(S)) structure in colloidal goethite dispersions. Furthermore, it displays a martensitic transition into the usual centered rectangular (R(C)) structure in an external magnetic field. The findings are rationalized in terms of entropic effects within a simple cell model. We interpret the results as an effect of the particle shape and the available degrees of freedom on the delicate balance between the space available for particle translations and rotations within the two structures.

Structural basis of myelin-associated glycoprotein adhesion and signalling.

Myelin-associated glycoprotein (MAG) is a myelin-expressed cell-adhesion and bi-directional signalling molecule. MAG maintains the myelin–axon spacing by interacting with specific neuronal glycolipids (gangliosides), inhibits axon regeneration and controls myelin formation. The mechanisms underlying MAG adhesion and signalling are unresolved. We present crystal structures of the MAG full ectodomain, which reveal an extended conformation of five Ig domains and a homodimeric arrangement involving membrane-proximal domains Ig4 and Ig5. MAG-oligosaccharide complex structures and biophysical assays show how MAG engages axonal gangliosides at domain Ig1. Two post-translational modifications were identified—N-linked glycosylation at the dimerization interface and tryptophan C-mannosylation proximal to the ganglioside binding site—that appear to have regulatory functions. Structure-guided mutations and neurite outgrowth assays demonstrate MAG dimerization and carbohydrate recognition are essential for its regeneration-inhibiting properties. The combination of trans ganglioside binding and cis homodimerization explains how MAG maintains the myelin–axon spacing and provides a mechanism for MAG-mediated bi-directional signalling.

Tetra- versus Pentavalent Inhibitors of Cholera Toxin.

The five B-subunits (CTB5) of the Vibrio cholerae (cholera) toxin can bind to the intestinal cell surface so the entire AB5 toxin can enter the cell. Simultaneous binding can occur on more than one of the monosialotetrahexosylganglioside (GM1) units present on the cell surface. Such simultaneous binding arising from the toxins multivalency is believed to enhance its affinity. Thus, blocking the initial attachment of the toxin to the cell surface using inhibitors with GM1 subunits has the potential to stop the disease. Previously we showed that tetravalent GM1 molecules were sub-nanomolar inhibitors of CTB5. In this study, we synthesized a pentavalent version and compared the binding and potency of penta- and tetravalent cholera toxin inhibitors, based on the same scaffold, for the first time. The pentavalent geometry did not yield major benefits over the tetravalent species, but it was still a strong inhibitor, and no major steric clashes occurred when binding the toxin. Thus, systems which can adopt more geometries, such as those described here, can be equally potent, and this may possibly be due to their ability to form higher-order structures or simply due to more statistical options for binding.

Chromium-modified goethite in an external magnetic field

The behaviour of the different liquid crystalline phases observed in colloidal dispersions of goethite has been studied by small angle x-ray scattering. The particles align parallel to a small magnetic field and perpendicular to a large magnetic field, which was observed in the isotropic and nematic phase. The smectic phase also aligned parallel to a small magnetic field but it formed an aligned columnar phase with a distorted hexagonal structure in higher magnetic fields. The columnar phase mainly aligned in higher magnetic fields. Goethite particles were successfully modified with chromium. The Cr-modified goethite particles show similar phase behaviour as normal goethite particles but with different magnetic properties. The relative importance of the permanent and induced magnetic moment was found to have changed.