De novo design and characterization of a family of metalloporphyrin proteins: The porphyrin assemblers

Abstract

De novo protein design utilizes computational and rational methods to produce proteins from pure principles. This approach critically tests our understanding of the basic principles underlying protein structure and function, while also enabling the design of structures with desirable properties that are not found in natural proteins. However, until now, de novo designed has been used primarily to reengineer proteins with structures and functions that closely resemble those found in natural proteins. We therefore explored computational and rational approaches to design and construct an asymmetric single chain protein bundle that self assembles and non-covalently binds a non-biological cofactor. Specifically incorporated into the scaffolds were interactions important for the function and stability of the bundles, including bis-His ligation of the non-biological cofactor, second shell hydrogen bonds, and favorable interactions between the cofactor and surrounding residues. Presented in this thesis are proteins that specifically recognize non-natural optically and electrically active cofactors. Briefly, Chapter Two focuses on the computational design of a protein that strongly resembles a natural four-helix bundle protein, but that recognizes a novel synthetic metalloporphyrin cofactor, the prototype protein, PAsc. PAsc was found to be monomeric, helical, and well folded, as demonstrated by size exclusion chromatography, AUC, CD, and NMR. PAsc also exhibits increased stability and helicity upon addition of this non-biological cofactor unique fold. Furthermore, computational and rational techniques were used to also change the prototype scaffold to examine the coordination of different metal cofactors and diversify the protein\u27s exterior, which is described in Chapters Four and Five. In a second application in Chapter Three, I demonstrate how the computational approach can be expanded to enable the design of a more novel protein-like scaffold that assembles porphyrins into linear arrays. The techniques used in Chapter Two are used here to demonstrate these arrays are tetrameric, well folded, and assemble around their designed number of cofactors. Prior to the work presented within, sequence-based design techniques had been used to design peptides that bind biological cofactors non-selectively. The design method provides control over the number, spacing, and chemical structure of the porphyrins, suggesting possible applications in the area of smart materials and nanotechnology