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.
Kajihara
Daisuke
D
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
Abe
Ryoji
R
Iijima
Issei
I
Komiyama
Chie
C
Sisido
Masahiko
M
Hohsaka
Takahiro
T
D002147:Calmodulin / Q000737:chemistry*; D002474:Cell-Free System; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D015394:Molecular Structure; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D011487:Protein Conformation; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D013329:Structure-Activity Relationship
D002147:Calmodulin; D005456:Fluorescent Dyes; C009722:4-aminophenylalanine; D010649:Phenylalanine