INVESTIGATION INTO THE ENZYMATIC FUNCTION OF AN EVOLVED, DE NOVO PROTEIN

Abstract

The Hecht laboratory has discovered that de novo proteins from a designed combinatorial library are able to rescue a series of E. coli auxotrophs, single-gene knockout strains unable to survive on minimal media. In particular, this thesis investigates synthetic protein 4F2, the product of directed evolution from an original, promiscuous auxotroph rescuer. 4F2 is able to efficiently rescue cells deleted for the fes gene, which encodes the protein ferric enterobactin esterase. In addition, 4F2 was found to facilitate cell growth by direct replacement of native esterase activity, as measured quantitatively by liquid chromatography and mass spectrometry (LC-MS) in vitro. However, the mechanism by which 4F2 performs this biochemical activity to enable life remains very much unknown. Investigation herein aimed to elucidate the sequence dependence of esterase activity of 4F2. To that end, 29 point mutants of 4F2 were constructed by single-site mutagenesis and tested for cell growth and color phenotype in Δfes cells. They were additionally screened for phenotype in the ΔfesΔfur double knockout strain and analyzed by LC-MS in vitro to determine their relative esterase activities, measured by cleavage of ferric enterobactin. Ultimately, tests of the 4F2 mutants demonstrate that mutations at several positions (V44, R85, and H74) severely compromise esterase activity, implicating these residues in catalytic function. If supplemented with knowledge of the structural active site of 4F2 and specific interactions between catalytic residues, this research offers tremendous promise for understanding how novel proteins are able to perform functions to sustain life in E. coli.