Real-time imaging of single-molecule fluorescence with a zero-mode waveguide for the analysis of protein-protein interaction.
Real-time imaging of single-molecule fluorescence with a zero-mode waveguide (ZMW) was achieved. With modification of the ZMW geometry, the signal-to-background ratio is twice that obtainable with a conventional ZMW. The improved signal-to-background ratio makes it possible to visualize individual binding-release events between chaperonin GroEL and cochaperonin GroES at a concentration of 5 microM. Two rate constants representing two-timer kinetics in the release of GroES from GroEL were measured with the ZMW, and the measurements agreed well with those made with a total internal reflection fluorescence microscopy. These results indicate that the novel ZMW makes feasible the direct observation of protein-protein interaction at an intracellular concentration in real time.
Miyake
Takeo
T
Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku, Tokyo 169-8555, Japan. miyake@ohdomari.comm.waseda.ac.jp
Tanii
Takashi
T
Sonobe
Hironori
H
Akahori
Rena
R
Shimamoto
Naonobu
N
Ueno
Taro
T
Funatsu
Takashi
T
Ohdomari
Iwao
I
D018835:Chaperonin 10; D018834:Chaperonin 60; D004867:Equipment Design; D008856:Microscopy, Fluorescence / Q000379:methods*; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods*
D018835:Chaperonin 10; D018834:Chaperonin 60