FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids.

We designed and synthesized new, fluorescent, non-natural amino acids that emit fluorescence of wavelengths longer than 500 nm and are accepted by an Escherichia coli cell-free translation system. We synthesized p-aminophenylalanine derivatives linked with BODIPY fluorophores at the p-amino group and introduced them into streptavidin using the four-base codon CGGG in a cell-free translation system. Practically, the incorporation efficiency was high enough for BODIPYFL, BODIPY558 and BODIPY576. Next, we incorporated BODIPYFL-aminophenylalanine and BODIPY558-aminophenylalanine into different positions of calmodulin as a donor and acceptor pair for fluorescence resonance energy transfer (FRET) using two four-base codons. Fluorescence spectra and polarization measurements revealed that substantial FRET changes upon the binding of calmodulin-binding peptide occurred for the double-labeled calmodulins containing BODIPY558 at the N terminus and BODIPYFL at the Gly40, Phe99 and Leu112 positions. These results demonstrate the usefulness of FRET based on the position-specific double incorporation of fluorescent amino acids for analyzing conformational changes of proteins.