Kaisa Kisko

Self-assembled films of hydrophobin proteins HFBI and HFBII studied in situ at the air/water interface.

Hydrophobins are a group of surface-active fungal proteins known to adsorb to the air/water interface and self-assemble into highly crystalline films. We characterized the self-assembled protein films of two hydrophobins, HFBI and HFBII from Trichoderma reesei, directly at the air/water interface using Brewster angle microscopy, grazing-incidence X-ray diffraction, and reflectivity. Already in zero surface pressure, HFBI and HFBII self-assembled into micrometer-sized rafts containing hexagonally ordered two-dimensional crystallites with lattice constants of 55 A and 56 A, respectively. Increasing the pressure did not change the ordering of the proteins in the crystallites. According to the reflectivity measurements, the thicknesses of the hydrophobin films were 28 A (HFBI) and 24 A (HFBII) at 20 mN/m. The stable films could also be transferred to a silicon substrate. Modeling of the diffraction data indicated that both hydrophobin films contained six molecules in the unit cell, but the ordering of the molecules was somewhat different for HFBI and HFBII, suggesting specific protein-protein interactions.

Thermodynamic and structural description of allosterically regulated VEGFR-2 dimerization.

VEGFs activate 3 receptor tyrosine kinases, VEGFR-1, VEGFR-2, and VEGFR-3, promoting angiogenic and lymphangiogenic signaling. The extracellular receptor domain (ECD) consists of 7 Ig-homology domains; domains 2 and 3 (D23) represent the ligand-binding domain, whereas the function of D4-7 is unclear. Ligand binding promotes receptor dimerization and instigates transmembrane signaling and receptor kinase activation. In the present study, isothermal titration calorimetry showed that the Gibbs free energy of VEGF-A, VEGF-C, or VEGF-E binding to D23 or the full-length ECD of VEGFR-2 is dominated by favorable entropic contribution with enthalpic penalty. The free energy of VEGF binding to the ECD is 1.0-1.7 kcal/mol less favorable than for binding to D23. A model of the VEGF-E/VEGFR-2 ECD complex derived from small-angle scattering data provided evidence for homotypic interactions in D4-7. We also solved the crystal structures of complexes between VEGF-A or VEGF-E with D23, which revealed comparable binding surfaces and similar interactions between the ligands and the receptor, but showed variation in D23 twist angles. The energetically unfavorable homotypic interactions in D4-7 may be required for re-orientation of receptor monomers, and this mechanism might prevent ligand-independent activation of VEGFR-2 to evade the deleterious consequences for blood and lymph vessel homeostasis arising from inappropriate receptor activation.

Structural analysis of vascular endothelial growth factor receptor-2/ligand complexes by small-angle X-ray solution scattering

Receptor tyrosine kinases play essential roles in tissue development and homeostasis, and aberrant signaling by these molecules is the basis of many diseases. Understanding the activation mechanism of these receptors is thus of high clinical relevance. We investigated vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), which regulate blood and lymph vessel formation. We analyzed the structural changes in the extracellular receptor domain that were induced by ligand binding and that represent the initial step in transmembrane signaling, culminating in the activation of the intracellular receptor kinase domain. High‐resolution structural information for the ligand binding domain became available recently, but the flexibility of the extracellular domain and inhomogeneous glycosylation of VEGFRs have prevented the production of highly diffracting crystals of the entire extracellular domain so far. Therefore, we chose to further investigate VEGFR structure by small‐angle X‐ray scattering in solution (SAXS). SAXS data were combined with independent distance restraint determination obtained by mass spectrometric analysis of chemically cross‐linked ligand/receptor complexes. With these data, we constructed a structural model of the entire extracellular receptor domain in the unbound form and in complex with VEGF.—Kisko, K., Brozzo, M. S., Missimer, J., Schleier, T., Menzel, A., Leppänen, V.‐M., Alitalo, K., Walzthoeni, T., Aebersold, R., Ballmer‐Hofer, K. Structural analysis of vascular endothelial growth factor receptor‐2/ligand complexes by small‐angle X‐ray solution scattering. FASEB J. 25, 2980–2986 (2011). www.fasebj.org

The relation between solution association and surface activity of the hydrophobin HFBI from Trichoderma reesei

Hydrophobins are small fungal surface active proteins that self‐assemble at interfaces into films with nanoscale structures. The hydrophobin HFBI from Trichoderma reesei has been shown to associate in solution into tetramers but the role of this association on the function of HFBI has remained unclear. We produced two HFBI variants that showed a significant shift in solution association equilibrium towards the tetramer state. However, this enhanced solution association did not alter the surface properties of the variant HFBIs. The results show that there is not a strong relationship between HFBI solution association state and surface properties such as surface activity.

Self-assembled structures of hydrophobins HFBI and HFBII

Hydrophobins are small proteins that function in the growth and development of fungi. The structures of class II hydrophobins HFBI and HFBII from Trichoderma reesei were studied using grazing incidence X-ray diffraction. HFBI was weakly ordered but HFBII formed a highly crystalline coating on water surface. Change from monoclinic to hexagonal structure was observed as the sample dried. The three-dimensional structures differed from the oblique two-dimensional structures observed in Langmuir-Blodgett monolayers of both HFBI and HFBII by atomic force microscopy.

Self-assembled films of hydrophobin protein HFBIII from Trichoderma reesei

Hydrophobins are a group of small amphiphilic proteins which are known to self-assemble on interfaces. They contain eight conserved cysteine residues, which make four disulfide bridges. A new hydrophobin protein, HFBIII, from the fungus Trichoderma reesei contains one extra cysteine residue, giving the protein a naturally reactive site. The self-assembly of hydrophobin protein HFBIII was studied using grazing-incidence X-ray diffraction and reflectivity. HFBIII self-assembles into a hexagonally ordered monolayer at an air/water interface and also forms crystalline coatings on a silicon substrate. The lattice constants for the hexagonal coatings are a = b = 56.5 A, γ = 120°. The self-assembled structure in the HFBIII film is very similar to those formed by two other T. reesei hydrophobins, HFBI and HFBII.

Structure of nickel nanoparticles in a microcrystalline cellulose matrix studied using anomalous small-angle X-ray scattering

Nickel nanoparticles were synthesized by adding aqueous nickel salt into a microcrystalline cellulose matrix. The NiII ions were reduced with either sodium borohydride, NaBH_4, or potassium hypophosphite, KH_2PO_2, in water or aqueous NH_3 medium. The mass fraction of Ni in the samples was between 3.7 and 8.9%. X-ray absorption spectra at the Ni K-edge showed that Ni was partially oxidized only in a sample reduced with NaBH_4. Wide-angle X-ray scattering results showed that nickel was in nanocrystalline or amorphous form in the samples. Upon heating fcc Ni, hcp Ni, NiO, Ni_3P and other Ni–P phases formed depending on the reduction parameters. Using anomalous small-angle X-ray scattering the nanometre-scale particle size distributions of the Ni particles were determined. A large fraction of particles less than 15 nm in size were observed in the samples reduced in aqueous ammonium compared with the samples reduced in water. Particles reduced in aqueous ammonium had a large ferromagnetic component.

Diffraction analysis of highly ordered smectic supramolecules of conjugated rodlike polymers

A small/wide-angle X-ray scattering and grazing incidence diffraction study of comb-shaped supramolecules of conjugated poly(2,5-pyridinediyl), acid dopant and hydrogen bonded amphiphilic side chains is reported. In solution, polymers are dissolved rodlike particles. When the side-chains are introduced, polymers self-assemble in hierarchic liquid crystals (LC). Diffraction patterns of aligned LC show h00, 020, and 004 reflections, and additional small-angle reflections along the polymer axis. A triangular correlation function indicating a very large correlation length is seen along the smectic axis. An aligned solid structure can be formed by cleaving side chains from the aligned LC.

Multiple orientation and nematic-hexagonal transition in uniaxially aligned polyfluorene thin films

We present a structural study of poly(9,9-bis(2-ethylhexyl)-fluorene-2,7-diyl) (PF2/6) in aligned thin films on a rubbed polyimide substrate. PF2/6 forms 5/2-helical hairy-rodlike molecular structure which is self-organized in the hexagonal structure. In thin films, the aligned rigid polymer chains are parallel to the substrate in the direction corresponding to the molecular backbone, c axis. The cells are flattened in the direction of the surface normal and -in particular- reveal a multiple orientation where the greater proportion of the crystallites have one crystal axis a perpendicular to the substrate surface but a small proportion are aligned with the crystal axis a parallel to surface. We find further that by reducing the polymer chain length to approximately twenty repeat units the degree of axial alignment of PF2/6 is considerably improved while the local order changes from hexagonal packing towards nematic phase with no sign of multiple orientation.