Fluorescence resonance energy transfer-based assay for DNA-binding protein tagged by green fluorescent protein.
Specific interaction between green fluorescent protein (GFP)-tagged human alpha- or gamma-enolase(97-242) (alpha or gammaENO(97-242)) and the rhodamine-labeled DNA fragment containing the c-myc P2 promoter was detected by a fluorescence resonance energy transfer (FRET)-based assay, designated as a "real-time FRET assay." The approach of donor (GFP) and acceptor (rhodamine) was caused by the association between ENO(97-242) and the c-myc P2 promoter, and the time-dependent increase in fluorescence intensity of the reaction mixture was observed at ex=400 nm and em=590 nm. The relative affinity (R(as)) of ENO(97-242) mutants to the wild type was investigated with a real-time FRET assay, and it was clarified that the amino acids that participated in the interaction existed comparatively broadly. Although it was difficult to measure the absolute value of the affinity for the binding protein by using this method, it was possible to investigate the relative affinity of mutants for the wild type. A real-time FRET assay using the GFP-tagged protein could be used as not only a qualitative, but also as a quantitative analysis, this being the best for investigating the key amino acids in binding proteins.
Aoki
Takashi
T
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Japan. aokit@hoku-iryo-u.ac.jp
Imamura
Tomoko
T
Ozaki
Hiroyuki
H
Ideuchi
Hideki
H
Tsuchida
Shirou
S
Watabe
Hiroyuki
H
D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D016259:Genes, myc / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012235:Rhodamines / Q000737:chemistry
D004268:DNA-Binding Proteins; D012235:Rhodamines; D049452:Green Fluorescent Proteins; D010751:Phosphopyruvate Hydratase