RAISE: Design of co-assembling peptides as recombinant protein fusion tags for integrating enzymes into supramolecular hydrogels

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This RAISE project is jointly funded by the Biological and Environmental Interactions of Nanoscale Materials program in CBET Division in the Engineering Directorate, the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences in the Biological Sciences Directorate, and the Office of Integrative Activities. In this multi-disciplinary project, nanomaterial scaffolds, or hydrogels, with immobilized enzymes will be produced using proteins as a design template. The proposed method provides a novel alternative to conventional immobilization techniques which are more difficult and time consuming. This novel assembly technique is designed so that it selectively self-assembles into nano fiber structures. This targeted design and assembly of proteins into nanofibers is an exciting new research frontier that is largely unexplored with regard to both fundamental protein assembly and design of functional nanomaterials. A multi-disciplinary team of investigators will combine their expertise in modeling, imaging and characterization methods in order to demonstrate novel protein assembly and to advance fundamental understanding of protein assembly. The proposed technique will be an extraordinary improvement over the current state-of-the-art in protein assembly methods. Successful implementation of the proposed technique is anticipated to have a long-term broad impact on the engineering of novel functional materials for various biomedical and biotechnological applications. A variety of educational activities will be developed, including an iPad app on protein folding, and a video describing how computational methods can be used to design new biomaterials.The proposed method bypasses conventional immobilization techniques in which the nanostructured scaffold is functionalized post-assembly via reactions at its surface. Instead, enzymes are expressed by bacteria from recombinant DNA with peptide fusion tags that mediate their assembly into nanostructured materials. The key to the method is the use of fusion tags that co-assemble into fibrillar structures when mixed with a complementary peptide, but remain unassembled when pure. Co-assembling peptides are an exciting research frontier that is virtually unexplored in both protein biophysics and functional nanomaterials. The goals of the project are to demonstrate novel enzyme-functionalized supramolecular hydrogels, and to advance fundamental biophysical understanding necessary to predict peptide co-assembly. Three specific aims are proposed: (1) characterize the assembly process and resultant structure for known â-sheet nanofiber-forming co-assembling peptide pairs, (2) design and test new pairs of selectively co-assembling peptides and, (3) test co-assembling peptides as tags to immobilize proteins in nanostructured biomaterials. Each aim will leverage the computational modeling expertise of PI Hall, with expertise in spectroscopic and microscopic characterization of peptide nanofibers provided by Co-PI Hudalla and solid-state nuclear magnetic resonance expertise provide by Co-PI Paravastu. The proposed enzyme immobilization method will be an extraordinary improvement over the current state-of-the-art in protein immobilization methods and hence opens the door to addressing long-standing challenges in installing biologically-active, folded proteins into biomaterials. A variety of educational activities will be developed, including an iPad app on protein folding, and a video describing how computational methods can be used to design new biomaterials.