Exploring non-natural cofactor binding and catalytic activities of de novo proteins

Abstract

Structural similarities and sequence homology within protein superfamilies are testaments to the limitation of Nature’s biochemical repertoire. Exploring beyond Nature’s boundaries might result in the discovery of new, and exciting catalytic activities. The Hecht Lab approaches this field by synthesizing non-natural, de novo proteins. An existing library of such proteins, called the ‘3rd Gen. Library’, was previously found to contain a high proportion of proteins which bind heme (Iron(II) Protoporphyrin IX) and catalyse biological reactions. This thesis aims to perform binding and reaction assays using proteins from the 3rd Gen. Library with unnatural cofactor Cobalt(III) Protoporphyrin IX (CoPPIX) instead of heme. Since CoPPIX is bound by heme-binding enzymes such as apo-cytochrome b562, it is hypothesized that the heme-binding 3rd Gen. Library proteins will also bind CoPPIX and perhaps even participate in catalysis. 96 proteins from the 3rd Gen. Library were studied for binding activity with CoPPIX and indeed several proteins were found to bind the cofactor. Then, the 96 proteins were assayed for catalytic activities. Redox-mediated Kemp Eliminase and Peroxidase activities were chosen as interesting, and feasible reactions to perform with the de novo proteins and CoPPIX. Reaction assays revealed a lack of Kemp Eliminase and Peroxidase activities of the 96 proteins with CoPPIX. However, the 96 proteins assayed represent a miniscule subset of the vast 3rd Generation Library and CoPPIX assisted catalytic activities might yet be shown by proteins not analyzed. This thesis promotes the study of de novo proteins and unnatural cofactors, such as CoPPIX, as it will provide insight into previously unexplored sequence space and enzymatic reaction mechanisms.