Electrochemical and functional studies of de novo alpha helical proteins from a designed combinatorial library

Combinatorial libraries of designed proteins facilitate the exploration of structures and functions of proteins which are unbiased by explicit rational design or natural evolution. This thesis examines functional and electrochemical properties of de novo designed alpha helical proteins from one such combinatorial library of 'binary patterned' proteins. The studies initiated in this thesis lay the foundation for the development of biosensors based on 'binary patterned' de novo proteins.; The proteins were immobilized onto gold surface using different strategies and were subjected to electrochemical and functional interrogation.; First, a four-helix bundle protein, S-824-C, was immobilized onto a gold electrode using organic linkers. The electrochemical response of heme-bound S-824-C to added N-donor ligands indicated that the protein scaffold modulates the binding of N-donor ligands to the buried heme in heme/S824C.; Second, de novo protein S-824-C was nanopatterned onto a gold surface in order to demonstrate the viability of preparing nanometer scale protein arrays.; Third, the 'binary patterned' de novo proteins were immobilized by exploiting the affinity between heme and the proteins and the suitability of the proteins was evaluated for developing hydrogen peroxide sensors. One of the proteins (n86) was shown to have half the activity of a horseradish peroxidase protein when both are immobilized. Further, it was observed that de novo proteins with less compact apo state showed better peroxidase activity in their heme bound form than proteins with more compact apo structure.; In a separate study, the phenylalanine residues in protein S-824 were mutated to create a site for binding small molecules and the presence of binding site was confirmed using computational analysis. Further, the protein S-824 and its mutant F64A were screened for binding to small molecules using saturation transfer difference (STD) NMR. Finally, the binding constants were determined using pulse field gradient spin echo (PFGSE) NMR experiments. This study showed that the F64A mutation in protein S-824 leads to the creation of a cavity that can bind small molecules specifically with high affinity.