earticle

영어

Single-molecule fluorescence resonance energy transfer (smFRET) analysis provides a variety of information including conformational and structural dynamics and localized or colocalized position of the tagged protein. For effective smFRET analysis of protein, site-specific dual-labeling with two fluorophores as an energy donor and an acceptor is crucial. We have shown that the site-specific labeling of protein via incorporation of an unnautural amino acid provides a greater contrast between the folded and unfolded states of the protein in smFRET analysis than the conventional labeling using double cysteines. As a model study, maltose-binding protein (MBP) was dually labeled via incorporation of ρ-azido-L-phenylalanine and cysteine at specific positions, immobilized on a surface, and subjected to smFRET analysis under native and denaturing conditions. The resulting histograms show that site-specific dual-labeling results in a more homogeneous distribution in protein populations. Considering this case of MBP, site-specific dual-labeling of the protein might offer a new chance for more precise smFRET analysis of the protein.