Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its -glycosylated N- and C-linker.

Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N- and C-termini.

A bio-inspired method for direct measurement of local wall shear rates with micrometer localization using the multimeric protein von Willebrand factor as sensor molecule.

Wall shear rates are critical for a broad variety of fluidic phenomena and are taken into account in nearly every experimental or simulation study. Generally, shear rates are not observable directly but rather derived from other parameters such as pressure and flow, often assuming somehow idealized systems. However, there is a biological system which is able to constantly measure the wall shear as a part of a regulatory circuit: The blood circulation system takes advantage of shear rate sensor (protein)molecules (multimeric forms of von Willebrand Factor, VWF), which are dissolved in the blood plasma and dramatically change their conformation under shear conditions.

COMPARATIVE PHYSIOCHEMICAL ANALYSIS OF HYDROPHOBINS PRODUCED IN ESCHERICHIA COLI AND PICHIA PASTORIS.

Hydrophobins (HFBs) are small surface-active proteins secreted by filamentous fungi. Being amphiphilic, they spontaneously form layers that convert surfaces from hydrophilic to hydrophobic and vice versa. We have compared properties of the class II HFB4 and HFB7 from Trichoderma virens as produced in Escherichia coli and Pichia pastoris.

Advanced portrayal of SMIL coating by allying CZE performance with in-capillary topographic and charge-related surface characterization.

A successive multiple ionic polymer layer (SMIL) coating composed of four layers improved the capillary electrophoretic separation of a recombinant major birch pollen allergen and closely related variants when poly(acrylamide-co-2-acrylamido-2-methyl-1-propansulfonate) (55% PAMAMPS) replaced dextran sulfate as terminal SMIL layer. 55% PAMAMPS decelerated the electroosmotic flow (EOF) due to its lower charge density. Atomic force microscopy (AFM) was used to investigate SMIL properties directly on the inner capillary surface and to relate them to EOF measurements and results of associated CZE separations of a mixture of model proteins and peptides that were performed in the same capillary.

Mapping molecular adhesion sites inside SMIL coated capillaries using atomic force microscopy recognition imaging.

Capillary zone electrophoresis (CZE) is a powerful analytical technique for fast and efficient separation of different analytes ranging from small inorganic ions to large proteins. However electrophoretic resolution significantly depends on the coating of the inner capillary surface. High technical efforts like Successive Multiple Ionic Polymer Layer (SMIL) generation have been taken to develop stable coatings with switchable surface charges fulfilling the requirements needed for optimal separation.

Shear-Dependent Interactions of von Willebrand Factor with Factor VIII and Protease ADAMTS 13 Demonstrated at a Single Molecule Level by Atomic Force Microscopy.

Vital functions of mammals are only possible due to the behavior of blood to coagulate most efficiently in vessels with particularly high wall shear rates. This is caused by the functional changes of the von Willebrand Factor (VWF), which mediates coagulation of blood platelets (primary hemostasis) especially when it is stretched under shear stress. Our data show that shear stretching also affects other functions of VWF: Using a customized device to simulate shear conditions and to conserve the VWF molecules in their unstable, elongated conformation, we visualize at single molecule level by AFM that VWF is preferentially cleaved by the protease ADAMTS13 at higher shear rates.

Ca(2+) concentration-dependent conformational change of FVIII B-domain observed by atomic force microscopy.

FVIII is a multi-domain protein organized in a heavy and a light chain, and a B-domain whose biological function is still a matter of debate. The 3D structure of a B-domain-deleted FVIII variant has been determined by X-ray crystallography, leaving unexplained the functional nature of the flexible B-domain which could play an important role in the structure-function relationship since it is removed during the activation process. To obtain clues on the function of the B-domain, the morphology of full-length FVIII and its isolated domains was determined in the absence or presence of Ca(2+).

Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate by covalent fusion to hydrophobins.

Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.

Cerato-platanins: a fungal protein family with intriguing properties and application potential.

Cerato-platanin proteins are small, secreted proteins with four conserved cysteines that are abundantly produced by filamentous fungi with all types of lifestyles. These proteins appear to be readily recognized by other organisms and are therefore important factors in interactions of fungi with other organisms, e.g.

Visualization of a protein-protein interaction at a single-molecule level by atomic force microscopy.

Atomic force microscopy is unmatched in terms of high-resolution imaging under ambient conditions. Over the years, substantial progress has been made using this technique to improve our understanding of biological systems on the nanometer scale, such as visualization of single biomolecules. For monitoring also the interaction between biomolecules, in situ high-speed imaging is making enormous progress.

Self-assembly at air/water interfaces and carbohydrate binding properties of the small secreted protein EPL1 from the fungus Trichoderma atroviride.

The protein EPL1 from the fungus Trichoderma atroviride belongs to the cerato-platanin protein family. These proteins occur only in filamentous fungi and are associated with the induction of defense responses in plants and allergic reactions in humans. However, fungi with other lifestyles also express cerato-platanin proteins, and the primary function of this protein family has not yet been elucidated.