Efficient single-molecule fluorescence resonance energy transfer analysis by site-specific dual-labeling of protein using an unnatural amino acid.
Single-molecule fluorescence resonance energy transfer (smFRET) measurement provides a unique and powerful approach to understand complex biological processes including conformational and structural dynamics of individual biomolecules. For effective smFRET analysis of protein, site-specific dual-labeling with two fluorophores as an energy donor and an acceptor is crucial. Here we demonstrate that site-specific dual-labeling of protein via incorporation of unnatural amino acid provides a clearer picture for the folded and unfolded states of the protein in smFRET analysis than 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, enabling a precise smFRET analysis of protein.
Moon-Hyeong
MH
Seo
Tae-Sun
TS
Lee
Eunkyung
E
Kim
Young Lag
YL
Cho
Hee-Sung
HS
Park
Tae-Young
TY
Yoon
Hak-Sung
HS
Kim
D000596:Amino Acids / Q000737:chemistry*; D002232:Carbocyanines / Q000737:chemistry; D002942:Circular Dichroism; D057930:Click Chemistry; D003545:Cysteine / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D058619:Maltose-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry
D000596:Amino Acids; D002232:Carbocyanines; D005456:Fluorescent Dyes; D058619:Maltose-Binding Proteins; C085321:cyanine dye 5; D010649:Phenylalanine; D003545:Cysteine