Protein-protein interface design has two challenging aspects: binding site identification and design of a de novo protein to scaffold the binding site interface. Currently very few if any protein-protein interfaces have been designed without first having an antibody structure(See related work below). I intend to change that with the following strategy.
binding site identification: Binding site (stub) identification is challenging because it requires design of polar protein-protein interfaces. If we look to nature for inspiration we see many protein-protein interfaces are in loops [Antibodies]. However, loop design in Rosetta is difficult. I intend to overcome this in 4 ways. First, I will design hydrogen bond networks in helical and loop stubs using a new protocol called hb-net developed by Scott Boyken. Second, I will develop a new technique that designs loops that are either structurally conserved in the PDB or whose loop direction can be forced by forming kinks early in the loop. Allowing loops in stub placement will dramatically increase the possible orientations for the polar residues, thereby increasing the chance we will find favorable hydrogen bond networks. Third, since the first two steps are computationally expensive I will utilize model-based search on high performance compute architectures to direct exploration toward the best stub locations. And fourth, since I know loops are still likely to be designed poorly I will produce multiple loop stubs from the same N and C stub termini. Having multiple loops for the same stub termini will increase the chances to find good loops without having to re-order the entire proteins through the use of a high-throughput chip assay.
de novo protein design : I intend to pursue two strategies to improve the design of functional de-novo proteins. The goal of both strategies is to design a topology that is able to scaffold a large binding site and also pass structure prediction filters.
Strategy 1. Repeat protein assembly : I propose to develop a protein design strategy that assembles proteins by connecting previously designed repeats with repeat junctions. Because all subunits already will have been experimentally tested it is very likely the new proteins will be viable experimentally. (See current work A)
Strategy 2. Broken chain assembly: I propose to develop a broken-chain assembly strategy that can connect multiple stubs together with a stabilizing backbone. Backbone assembly will iterate between backbone design and identification of poorly designed regions which will be identified by a fast method to assess if a protein folds computationally.
Both strategies offer a way to quickly design large functional de-novo proteins that pass computational foldability tests.