Ligand-Actuated Fluorescent Protein Biosensors

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

Molecular detection is a pervasive challenge in biology, medicine, and the environmental sciences. Most often, the detection process is mediated by antibodies or other binding moieties that specifically recognize the target molecules. Subsequently, the target is imaged or detected by a secondary means such as fluorescence or enzymatic reaction. Recently, we and others have engineered fluorescence proteins endowed with binding capability that allow facile one-step detection of target ligands as a result of their intrinsic fluorescence properties. While a suitable substitute for antibodies or other binding scaffolds, the true potential in a fluorescent binding protein would be if the fluorescence generated were a function of the target ligand presence. In other words, such a ligand-actuated fluorescent protein biosensor would undergo reconstitution or changes in the chromophore upon binding to cognate ligand yielding a fluorescence readout. Here we endeavor to create an ideal ligand-actuated fluorescence protein platform that is compatible with broad library-based identification approaches, analogous to those used in antibody engineering, capable of yielding ligand-actuated fluorescent proteins against a wide range of molecular targets. In addition to the research objectives, this proposal possesses significant broader impact. First, it provides a new conceptual framework and toolkit for the creation of ligand-responsive biosensors. In addition, ligand-responsive biosensors could revolutionize the study of processes in living cells and the facile detection of contaminants or toxic materials. Next, the project is designed to train a graduate student and several undergraduate students in the protein engineering field to prepare them for careers in industry and academia. To date, the PI has trained 33 undergraduate and graduate students, several of whom are from underrepresented backgrounds. Research results will be integrated into courses that the PI regularly teaches as protein engineering modules in his "Design of Biological Molecules" graduate course and as coupled transport-reaction problems in his undergraduate mass transfer course. Finally, research results will also be disseminated to high school students in the form of the Wisconsin High School State Science Olympiad, through an "Applied Protein Modeling for Medical Uses" symposium that the PI organizes and hosts.