Session: Current Topics in Biological Chemistry:

Acridonylalanine (Acd) is a fluorescent amino acid that can be a valuable tool for studying protein dynamics. 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 (PET) probe pair. We have reported successful, selective genetic incorporation of Acd by an aminoacyl tRNA synthetase to site-specifically label a protein of interest expressed in E. coli and/or mammalian cells. This has allowed us to monitor protein localization using confocal microscopy and fluorescence lifetime imaging microscopy (FLIM). We have also developed new Acd derivatives that can shift Acd’s fluorogenic properties while still being ribosomally permitted for genetic incorporation. One derivative has been shown to be an enhanced FRET acceptor in a peptide cleavage assay and can be activated by the existing Acd tRNA synthetase for genetic incorporation. We are working to evolve aminoacyl tRNA synthetases to improve the incorporation of new Acd derivatives. 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.

Prof. James James Petersson

University of Pennsylvania

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