Protein Design and Engineering Using the Fluorescent Non-canonical Amino Acid L-(7-hydroxycoumarin-4-yl)ethylglycine
Proteins are, arguably, the most complicated molecular machines found in nature. From the receptor proteins that decorate the exterior of cell membranes to enzymes that catalyze the slowest of chemical reactions, proteins perform a wide variety of essential biological functions. A reductionist view of proteins as a macromolecular group, however, may hold that they simply interact with other chemical species. Notably, proteins interact with other proteins, other biological macromolecules, small molecules, and ions. This in turn makes proteins uniquely qualified for use technological use as sensors of said chemical species (biosensors). Several methods have been developed to convert proteins into biosensors. Many of these techniques take advantage of fluorescence spectroscopy because it is a fast, non-invasive, non-destructive and highly sensitive method that also allows for spatiotemporal control. This, however, requires that first a fluorophore be added to a target protein. Several methods for achieving this have been developed from large, genetically encoded autofluorescent protein tags, to labeling with small molecule fluorophores using bioorthogonal chemical handles, to genetically encoded fluorescent non-canonical amino acids (fNCAA). In recent years, the fNCAA, L-(7-hydroxycoumarin-4yl)ethylglycine (7-HCAA) has been used in to develop several types of biosensors.
The dissertation I present here specifically addresses the use of the fNCAA L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) in protein-based biosensors. I demonstrate 7-HCAA’s ability to act as a Förster resonance energy transfer (FRET) acceptor with tryptophan as the FRET donor in a single protein containing multiple tryptophans. I the describe efforts to elucidate—through both spectroscopic and structural characterization—interactions within a 7-HCAA containing protein that governs 7-HCAA fluorescence. Finally, I present a top-down computational design strategy for incorporating 7-HCAA into proteins that takes advantage of previously described interactions. These reports show the applicability of 7-HCAA and the wider class of fNCAAs as a whole for their use of rationally designed biosensors.