Evaluating insertion tolerance of the MtrC protein to design biosensors for detecting endocrine disruptors in Shewanella oneidensis

Abstract

Endocrine-disrupting chemicals are environmental contaminants that interfere with hormone signaling and pose significant risk to human and ecological health, even at low concentrations. This work explores the development of electrochemical biosensors for real-time, on-site detection of endocrine-disrupting chemicals by transduction of ligand binding into measurable electrical output. Specifically, steroid hormone receptor ligand-binding domains are inserted along the MtrC protein sequence, the terminal reductase of the MtrCAB electron transfer pathway, thus creating a protein switch that enables ligand-dependent control of extracellular electron transfer. This thesis presents two complementary approaches for designing protein switches. First, insertion sites were rationally chosen based on MtrC sequence conservation and structural analysis. Initial experimental characterization revealed at least one functional switch: the estrogen receptor insertion at position 323 produced ligand-dependent modulation of electron transfer in response to 4-hydroxytamoxifen and 4-nonylphenol. Second, for a high-throughput approach, I developed an MtrC library with insertions at all amino acid positions to determine tolerance to domain insertions and identify sites that preserve electron transfer functionality. Through these approaches, this work establishes a framework for engineering electrochemical protein switches and provides both experimental validation for rational design and a scalable strategy for more comprehensive screening.