Strategy for efficient site-specific FRET-dye labeling of ubiquitin.
To study conformational changes within a single protein molecule, sp-FRET (single pair fluorescence resonance energy transfer) is an important technique to provide distance information. However, incorporating donor and acceptor dyes into the same protein molecule is not an easy task. Here, we report a strategy for the efficient double-labeling of a protein on a solid support. An ubiquitin mutant with two Cys mutations, one with high solvent accessibility and the other with low solvent accessibility, was constructed. The protein was bound to magnetic beads and reacted with the dyes. The first dye reacted with the side-chain of the Cys with the high solvent accessibility and the second with the other Cys under partially denaturing conditions. Using this method, we can easily label two dyes in a site-specific way on ubiquitin with a satisfied yield. The labeling sites for donor and acceptor dyes can be easily swapped.
Kao
Michael Wen-Pin
MW
Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, R. O. C.
Yang
Li-Ling
LL
Lin
Jacky Chih-Kai
JC
Lim
Tsong-Shin
TS
Fann
Wunshain
W
Chen
Rita P-Y
RP
D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism
D005456:Fluorescent Dyes; D025801:Ubiquitin