The aim of this proposal is the design and production of engineered proteins for nanotechnology, biomaterials and sensor applications. Our objective is to develop a set of molecular components allowing the systematic assembly of functional nanostructures constructed of molecular "struts" and "nodes". Struts are molecular components that basically function as linear structural elements. Nodes are molecular connectors that have multiple attachment points with defined geometry. The combination of struts and nodes results in the production of 2 and 3-D lattices that can have utility themselves and/or can be further functionalized through chemical modification or the incorporation of additional specific binding proteins. Our initial strut molecule is streptavidin, a tetrameric protein molecule of MW 60,000 with D2 symmetry that has 4 high-affinity binding sites for the vitamin biotin. In the strut application, each streptavidin tetramer will form 2 biotin-binding interactions with a node protein. The initial objective of the current proposal is to design and produce two node proteins: one having three-fold symmetry to allow the formation of self-organizing 2D hexagonal lattices, and a node with 4-fold symmetry allowing formation of self-organizing 2D square lattices. We have selected candidate node molecules from known crystal structures of proteins derived from thermostable bacteria, and propose to use protein engineering approaches to a.) introduce sites that can be functionalized with biotin for connection with the strut protein streptavidin b.) introduce anchoring sequences (e.g. poly-histidine sequences) and/or sites that can be functionalized with other chemical molecules (e.g. lipids or thiols) to allow oriented immobilization on 2D surfaces, and c) create single chain variants with reduced binding site symmetry that can be used to sequentially assemble structurally complex nanostructures. We intend to design the molecular components and the encoding genes at our company Imiplex, to produce them through a contract biotechnology company, and to evaluate them through our own resources and collaborative relationships with university research laboratories. We intend to make these materials widely available to the biomedical, biotechnology and nanofabrication communities through a direct web-based sales process. Availability of these materials will facilitate the development of a new generation of functional nanodevices, biosensors, and biomaterials.
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