Hinge motion protein-based fluorescence assays for biosensing and biomolecular screening application

The use of proteins for both biosensing systems and components of modern high-throughput screening (HTS) assays has emerged from the advances in genetic, microbiological and biochemical technologies. There are two basic strategies that have been used to develop protein-based detection systems. The first is to find a protein with a specific binding capability and engineer a signal transduction function into it, and the second, to find a protein with a known signal transduction function and engineer it to contain the appropriate binding sites. The aim of this work is primarily to make use of the first strategy and to integrate the biological specificity of two well-characterized binding proteins into useful assays for the detection of pharmaceutically relevant biomolecules. This integration can be achieved by coupling the appropriate signal transduction functions to proteins with known natural binding characteristics. The intrinsic binding capacities of two proteins: the E. coli galactose/glucose binding protein and calmodulin have been utilized in the described studies. Each protein can exist as two or more structurally unique states with separate binding affinities that are in equilibrium with each other. This is due to differences in their local and global conformational arrangements. The presented studies make use of "fluorescent protein biosensors", which are fluorescently labeled proteins designed to detect and report changes in their environment. Chapter one provides a detailed background covering bacterial periplasmic binding proteins used as biorecognition elements and also calmodulin and the green fluorescent protein, which have been used in the studies contained herein. Chapters two and three detail studies using competitive fluorescence resonance energy transfer for the detection of calmodulin antagonists. Chapters four and five comprise two different methods for developing fluorescent protein indicators for glucose utilizing the E. coli galactose/glucose binding protein. The application of these fluorescent reporters as reagents in strategies aimed at overcoming some current challenges in biopharmaceutical assay development is described.;Keywords. Hinge-motion binding proteins, High throughput screening assays, Glucose sensing, Protein-based fluorescence assays, Chimeric proteins.