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Updated: Jun 12, 2026

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy
Published on: May 12, 2020
Single-molecule study on the decay process of the football-shaped GroEL-GroES complex using zero-mode waveguides
Tomoya Sameshima1, Ryo Iizuka, Taro Ueno
1Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Abstract:
It has been widely believed that an asymmetric GroEL-GroES complex (termed the bullet-shaped complex) is formed solely throughout the chaperonin reaction cycle, whereas we have recently revealed that a symmetric GroEL-(GroES)(2) complex (the football-shaped complex) can form in the presence of denatured proteins. However, the dynamics of the GroEL-GroES interaction, including the football-shaped complex, is unclear. We investigated the decay process of the football-shaped complex at a single-molecule level. Because submicromolar concentrations of fluorescent GroES are required in solution to form saturated amounts of the football-shaped complex, single-molecule fluorescence imaging was carried out using zero-mode waveguides. The single-molecule study revealed two insights into the GroEL-GroES reaction. First, the first GroES to interact with GroEL does not always dissociate from the football-shaped complex prior to the dissociation of a second GroES. Second, there are two cycles, the "football cycle " and the "bullet cycle," in the chaperonin reaction, and the lifetimes of the football-shaped and the bullet-shaped complexes were determined to be 3-5 s and about 6 s, respectively. These findings shed new light on the molecular mechanism of protein folding mediated by the GroEL-GroES chaperonin system.
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