Designing Functional Metalloproteins

Metalloproteins are critical cellular workhorses, catalyzing difficult but essential transformations that are required for functions as diverse as energy generation, metabolism, or signaling. The interplay between protein structure and catalytic activity of these enzymes forms the foundation of our work. The protein may influence both the first

coordination sphere (ligand type, number, and relative geometry) as well as the outer coordination environment. Despite the indispensable nature of this chemistry, a fundamental understanding of the relationship between metal ion catalysts and a protein scaffold remains elusive. While native metalloenzyme studies provide valuable

insights, interpretations of these systems are often inadequate because of the intricacy of the scaffold or the presence of multiple metal centers. Similarly, small molecules can afford insight on reactions but are often limited by the solvent or synthetic complexity. For this reason, we utilize an intermediate approach with de novo design

of -helical proteins which are easily synthesized, water soluble, recapitulate native metalloprotein active sites well, and provide native-like folds that can be manipulated simply in order to limit the variables that are probed during our studies. Our long-term goal is to describe to full chemical resolution the processes by which metals

interact with three stranded coiled-coils (3SCC) composed of -helical proteins in order to achieve specific metal structures and afford the desired catalysis. The overall objective is to insert metals into well-defined scaffolds that can then be altered to test important hypotheses that will explain how these catalytic centers work. Our

central hypothesis is that well-defined de novo metalloproteins can be used to interrogate metal behavior within designed proteins in ways that would be difficult to achieve with native proteins or small molecules. Our basic premise is that de novo protein design provides simple, highly-controllable, and easily modified scaffolds that

are well suited to extract fundamental information on metal chemistry. These structures provide a framework to examine and compare systematic perturbations to different coordination sites in aqueous peptidic environments, which is often difficult to do with small molecule models, especially those insoluble in water. The rationale of the

proposed research is that it will provide new information on protein-metal interactions that have eluded the

scrutiny of other approaches due to the complexity or insolubility of the natural systems. These studies will eventually

form the basis for developing new catalysts with different functions using essentially the same protein ligands but differentmetals. We can also now explore the influence of asymmetric first and second coordination sphere environments and changing the

helical twist on the structural and catalytic behavior of proteins. Our hypothesis will be tested through three Specific Aims: 1) Evaluation of catalytic activity for metal centers within existing 3SCC; 2) Assessing for the first time modifications to catalytic activity caused by insertion of sequence discontinuities; and 3) Using our breakthrough

preparation of heavy metal templated heterotrimeric 3SCCs to generate asymmetric metal catalysts. This research is significant and innovative as it identifies new approaches to optimize biomolecular catalysis.