Rational design of three-dimensional protein assemblies using non-canonical amino acids

About This Grant

NON-TECHNICAL SUMMARY: The ability to precisely design and assemble three-dimensional biomaterials will open the door for significant impacts in many fields including biotechnology, bioremediation, drug delivery, and advanced medical technologies. To this end, this project will develop, validate, and apply novel approaches for the design and precise assembly of proteins, containing non-natural amino acids, into three-dimensional frameworks to create new classes of functional biomaterials. The proposed approach promises to be readily extensible to a variety of systems and applications, thereby making it an exciting new method for biomaterials design. In addition, this proposal includes research and teaching activities in biochemistry, structural biology, protein engineering, and chemical biology that provide unique opportunities for training highly interdisciplinary students and broadening participation in the future STEM workforce of the United States. TECHNICAL SUMMARY: The primary goal of the proposed research is to develop a general method for the assembly of large, ordered 3D protein biomaterials. Standard protein crystals are typified by properties including close packing between protein partners and a relative inability to design or control the lattices in which the assemblies form. The ability to control the geometric relationships and distances between assembled proteins within the lattice would allow for the development of a new class of ordered 3D protein materials; at present, this degree of control over assembly is not possible. This work proposes a novel approach for generating “Coordinated Protein Frameworks” (CPFs), which represent extended protein lattices with defined geometries and tunable distances between each of the proteins in the lattice. The approach leverages non-canonical amino acids (ncAAs), whose side chains include chemical functional groups not present in the twenty standard amino acids. ncAAs containing “bioorthogonal” chemical functional groups will be incorporated at specified positions in a target protein that will define the symmetric arrangement between proteins in the resulting lattice. CPF assembly will then be initiated using small molecule linkers that terminate in chemical functional groups that will specifically react with the ncAAs and will not cross react with any natural amino acids. The symmetries of the CPF assemblies and the “pore sizes” of the lattices can both be readily tuned by altering the position of ncAA incorporation and modulation of the linker length, respectively. This novel approach promises to create a new class of tunable protein biomaterials that are similar to well-characterized small molecule systems like metal organic frameworks. Furthermore, because of the generality of the approach, essentially any protein (or potentially multiple proteins) could be patterned in three-dimensional space in an unprecedented way. The efforts of this study will provide new insights into engineered protein self-assembly processes and guide further research efforts on biomolecular and biomaterial design. Finally, this proposal includes educational activities in biochemistry, structural biology, protein engineering, and chemical biology that provide unique opportunities for training highly interdisciplinary students and broadening participation in the future STEM workforce of the United States. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.