TOWARD FUNCTIONAL DE NOVO PROTEINS: SEARCHING FOR KEMP ELIMINATION CATALYSIS AND EMBELIN BINDING IN DESIGNED COMBINATORIAL LIBRARIES

Abstract

Designing proteins with novel functions is the ultimate test of scientific understanding of the physical principles underlying protein structure and function, and de novo proteins with novel biochemical functions have important potential applications in medicine and industry. The Hecht Lab uses computational and combinatorial methods to design libraries of de novo proteins biased toward stable four-helix bundle structures. The present work investigates whether proteins designed by the Hecht Lab can catalyze chemical reactions and bind small molecules. In particular, de novo proteins are assayed for catalysis of the Kemp elimination, a well-studied model system for enzyme design, and for binding of embelin, a natural product that inhibits XIAP. In a screen of 1208 library proteins in lysate, 71 proteins showing a Kemp elimination reaction rate above background were identified. However, the high Kemp elimination reaction rate in empty vector lysate, the intended negative control, precluded meaningful analysis of test results in lysate. S824, a de novo protein with a previously determined structure, was found to exhibit Kemp elimination activity in lysate but not in pure form; similarly negative results were found for other library proteins. However, a purified protein from the 7X mutant sub-library was found to exhibit catalytic activity in the Kemp elimination. Several proteins from the R4HB library were found to increase the solubility of embelin, with binding constant determination ongoing. Overall, these results suggest that a combination of combinatorial and computation methods may enable catalysis and small molecule binding; however, the design of functional de novo proteins remains a complex challenge.