The Development of Intrinsically Fluorescent Unnatural Amino Acids for In Vivo Incorporation into Proteins

Abstract

The amino acid acridonylalanine (Acd) can be a valuable tool for studying protein dynamics. On its own, Acd's long fluorescence lifetime of 15 ns has been beneficial for polarization experiments to reveal protein binding dynamics in biological pathways. Acd can also reveal structural dynamics as part of a Förster resonance energy transfer (FRET) or photoinduced electron transfer (eT) probe pair. We have reported successful, selective genetic incorporation of Acd by an aminoacyl tRNA synthetase. This allows us to site-specifically label a protein of interest expressed in E. coli with minimal perturbation. While Acd is valuable in itself, we are developing on a series of Acd derivatives that can shift Acd's fluorogenic properties while still being ribosomally permitted for genetic incorporation. We have shown that we can red shift Acd's absorption and emission spectra and modulate it's brightness. One derivative has been shown be an enhanced FRET acceptor in a peptide cleavage assay and can be activated by the aminoacyl tRNA synthetase for genetic incorporation. Our long-term goal is to develop FRET pairs that can be completely genetically encoded to enable monitoring of protein processes that cannot be accessed with other labeling methods, such as real-time monitoring of co-translational protein folding.