| Biologically active surfaces that are capable of specific recognition and binding processes are of crucial importance for the development of a new generation of cell and protein arrays for drug discovery, diagnostic assays and biosensor devices. In most cases cell-membrane associated recognition processes are governed by protein-protein or peptide-protein interactions. In order to mimic these natural interactions, several requirements have to be fulfilled: a) proteins have to be immobilized in their native form on the surface, b) the possibility of multiple protein-protein interactions should be allowed and c) control over spatial positioning is mandatory. One of the most extensively studied and easily applied methods for the construction of well defined nano- to micropatterns on surfaces is phase separation of block copolymers. However, the introduction of a functional scaffold to which proteins can be attached without causing denaturation is more difficult to achieve with this patterning technique. A method that allows proteins to be immobilized in their active conformation is the use of a polypeptide scaffold in between the protein and the substrate. A further advantage of applying polypeptide moieties is that the information stored in the primary amino acid sequence can efficiently be translated in well-defined 3-D organization and functionality. By combining polypeptide elements with synthetic polymers into a hybrid block copolymer both patterning and immobilization capability could be introduced simultaneously. Recently, we have reported a novel approach toward block copolymers in which a designed, recombinantly produced protein polymer is combined with the synthetic polymer poly ethylene glycol (PEG). The protein polymer block consists of tandem repeats of the amino acid sequence -(AG)3EG- (A= alanine, G = glycine, E = glutamic acid). This polypeptide folds into well-defined beta-sheet crystals with the glutamic acid residues confined to the crystal surface. Control over the hierarchical assembly process of these beta-sheet structures is obtained by preparation of triblock copolymers consisting of the beta-sheet polypeptide as central block in combination with two PEG end blocks. The attachment of the flanking PEG chains blocks the formation of plate-like crystals and results in the assembly into well-defined fibrils, which have a natural tendency to align and form regular patterns on a surface. Since the polypeptide structure is constructed by means of protein engineering, absolute control over amino acid sequence and, hence, the properties of the beta-sheet fiber can be obtained, with respect to width, height, and surface functionality. Fiber dimensions can be varied in the nanometer range, functional groups can be positioned at the beta-turns with 5Å precision. It is the aim of the research described in this proposal to utilize the high level of control over chemical structure and 3-D organization of these materials to create generic nanometer sized scaffolds for attachment of functional moieties. These scaffolds will be applied for the production of biologically active surfaces. In order to achieve nanopatterned bioactive surfaces the following elements have to be investigated: i) synthesis of the block copolymers: a series of block copolymers will be made in which the length of both the -sheets and the PEG chains will be varied. ii) improvement of alignment of the beta-sheet fibers: The fibers will be subjected to film casting and electric forces in order to reach high levels of alignment, which is necessary for patterning. iii) functionalization of the beta-sheet fibers with ligands or recognition domains, and binding studies: surfaces will be modified with biotin and a peptide epitope derived from the Malaria parasite, in order to investigate binding studies with streptavidin and antibodies respectively. iv) creating fibers capable of binding multiple proteins: diblock polypeptides will be constructed by protein engineering, in which one half contains glutamic acids at the turn positions and t he other half acetylene functional amino acids. This allows patterning of 2 different bioactive moieties in close proximity of each other. The proof of principle of this approach will be tested with FRET analysis. |
