Single-molecule Study on the Decay Process of the Football-shaped GroEL-GroES Complex Using Zero-mode Waveguides

Tomoya Sameshima,Ryo Iizuka,Taro Ueno,Junichi Wada,Mutsuko Aoki,Naonobu Shimamoto,Iwao Ohdomari,Takashi Tanii,Takashi Funatsu

doi:10.1074/jbc.m110.122101

Tomoya Sameshima, Ryo Iizuka + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m110.122101

Copy DOI

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.

Highlights

The widely accepted model of protein folding mediated by the GroEL-GroES chaperonin system is as follows. (i) The denatured protein is injected into one of GroEL rings upon the ATP-dependent formation of the GroEL-GroES complex. (ii) ATP hydrolysis in the cis-ring results in the formation of the GroEL-GroES complex with bound ADP. (iii) Subsequent ATP binding to the opposite ring induces the release of GroES, ADP, and the encapsulated protein from the cis-ring. (iv) The trans-ring is reoriented to a new cis-ring, thereby allowing the ATPase cycle (1–5)
To characterize the formation processes of the football-shaped complexes, we picked up the events that signals from Cy3-labeled t-ES98C (Cy3-ES) were detected while Cy5-labeled GroEL (Cy5-EL) molecules stayed on the surface of zero-mode waveguides (ZMWs)
Surface-immobilized A488bio-ES was immersed in a solution containing 50 nM Cy5-EL, 300 nM Cy3-ES, 2 mM ATP, and excess amounts of LA, in which the disulfide bonds were reduced. 300 nM Cy3-ES was required to obtain saturating amounts of the football-shaped complexes

Summary

Introduction

The widely accepted model of protein folding mediated by the GroEL-GroES chaperonin system is as follows. (i) The denatured protein is injected into one of GroEL rings (cis-ring) upon the ATP-dependent formation of the GroEL-GroES complex. (ii) ATP hydrolysis in the cis-ring results in the formation of the GroEL-GroES complex with bound ADP. (iii) Subsequent ATP binding to the opposite ring (trans-ring) induces the release of GroES, ADP, and the encapsulated protein from the cis-ring. (iv) The trans-ring is reoriented to a new cis-ring, thereby allowing the ATPase cycle (1–5). Decay Process of the Football-shaped GroEL-GroES Complexes Labeled GroEL and GroES exhibited behaviors similar to those of the wild-type proteins (supplemental Fig. S1).

Results

Conclusion