The De Novo Design of Protein-Protein Interfaces

The primary goal of this research project was to explore the biophysical forces that drive biomolecular complex formation and further elucidate the parameters that give rise to specific protein/protein interactions. Thus far a large body of data has been collected on natural protein complexes and it was our intention to add to this body by engineering de novo (from scratch) protein/protein complexes. Using this inverse approach we have furthered the understanding of biomolecular specificity by engineering protein complexes from previously monomeric test proteins. The goal is to elucidate the key physical features needed to drive specific protein/protein interactions. It is considered inverse because, instead of studying natural complexes, we are exploring key physical parameters by designing novel protein/protein interfaces. To achieve this we initially utilized robust computational algorithms but then ultimately succeeded by combining these with rational design methods based primarily on the X-ray crystal structures of early imperfect results. Using these methods we recently succeeded at driving small test proteins to form complexes of specific structure. After a number of tantalizing yet imperfect results we ultimately achieved success by designing metal binding sites at the interface of the target dimer complexes. These relatively small complexes were generated using the small test protein, protein-G (56 amino acids), as a design scaffold. The subunits of our designed complexes are perfectly monomeric in the absence of metal, yet form very clean dimers in the presence of zinc ions. We are in the process of designing and testing additional mutants with the goal of further exploring the important biophysical features that drive these novel bimolecular dimer complexes.