Comparison of FArster-Resonance-Energy-Transfer Acceptors for Tryptophan and Tyrosine Residues in Native Proteins as Donors

• Published in: Journal of fluorescence Vol. 23; no. 1; pp. 147 - 157
• Main Authors: zhang, Yi, Yang, Xiaolan, Liu, Lin, Huang, Zuexia, Pu, Jun, Long, Gaobo, Zhang, Ling, Liu, Dong, Xu, Bangtian, Liao, Juan, Liao, Fei
• Format: Journal Article
• Language: English
• Published: 01.01.2013
• Subjects: Buffers; Excitation; Fretting; Phosphates; Proteins; Residues; Tryptophan; Tyrosine
• ISSN: 1053-0509; 1573-4994
• DOI: 10.1007/s10895-012-1128-z

Homogenous bioaffinity analysis with tryptophan/tyrosine residues in native proteins as FArster-resonance-energy-transfer (FRET) donors is feasible when suitable fluorophors can act as FRET acceptors in ligands (FRET probes) and FRET efficiency in complexes of proteins and FRET probes is high enough. In complexes of proteins and FRET probes, suitable acceptors should have excitation peaks around 335 nm and high rotation freedom, are preferred to have sufficient quantum yields and excitation valleys around 280 nm. In protein binding sites mimicked with mixtures of neutral phosphate buffer and organic solvents, quantum yields of candidate acceptors are altered inconsistently but their excitation peaks show tiny changes. Fluorophores as acceptors in such FRET probes are buried inside glutathione-S-transferase and have low rotation freedom, but are localized on streptavidin surface and display high rotation freedom; FRET efficiency in complexes of streptavidin and its FRET probes is much stronger than that in complexes of glutathione-S-transferase and its FRET probes. Specially, the quantum yield is about 0.70 for free 1-naphthylamine probe in neutral phosphate buffer, about 0.50 for 1-naphthylamine probe bound by streptavidin, and about 0.15 for that bound by glutathione-S-transferase. The quantum yield is about 0.06 for free dansylamide probe, about 0.11 for dansylamide probe bound by streptavidin and about 0.27 for that bound by glutathione-S-transferase. Therefore, 1-naphthylamine and dansylamide are effective acceptors when they localize on surfaces of complexes of proteins and FRET probes.