Fig. 2: Three-color FRET in protein folding and binding.

a Three different fluorophores are attached to α₃D site-specifically. Alexa 488 (donor, D) and Alexa 594 (acceptor 1, A1) are attached to 4-acetylphenylalanine (UA) at the N-terminus and cysteine at residue 33, respectively. Then, a cysteine residue is attached to the C-terminus using sortase-mediated ligation of a short peptide GGGC. This cysteine residue is labeled with CF680R (acceptor 2, A2). The labeled protein is immobilized on a polyethyleneglycol (PEG)-coated glass surface using biotin-streptavidin linkage. In two-color FRET, site-specific labeling of two fluorophores is not usually necessary because the two species with the different donor and acceptor positions would not cause a significant difference in the FRET efficiency unless fluorescence quenching occurs at one of the two labeling positions. In three-color FRET, however, when A1 and A2 are switched, for example, the distances between D and A1 and between D and A2 become different. b D and A1 are attached to 4-acetylphenylalanine and cysteine residues at the N- and C-termini of the transactivation domain (TAD), respectively, which is immobilized on the surface. TAD is incubated with A2-labeled NCBD in solution at a concentration close to the dissociation constant to monitor binding and dissociation events.