GroEL mediates protein folding with a two successive timer mechanism.
GroEL encapsulates nonnative substrate proteins in a central cavity capped by GroES, providing a safe folding cage. Conventional models assume that a single timer lasting approximately 8 s governs the ATP hydrolysis-driven GroEL chaperonin cycle. We examine single molecule imaging of GFP folding within the cavity, binding release dynamics of GroEL-GroES, ensemble measurements of GroEL/substrate FRET, and the initial kinetics of GroEL ATPase activity. We conclude that the cycle consists of two successive timers of approximately 3 s and approximately 5 s duration. During the first timer, GroEL is bound to ATP, substrate protein, and GroES. When the first timer ends, the substrate protein is released into the central cavity and folding begins. ATP hydrolysis and phosphate release immediately follow this transition. ADP, GroES, and substrate depart GroEL after the second timer is complete. This mechanism explains how GroES binding to a GroEL-substrate complex encapsulates the substrate rather than allowing it to escape into solution.
Ueno
Taro
T
Department of Physics, School of Science and Engineering, Waseda University, 3-4-1 Okubo, Tokyo 169-8555, Japan.
Taguchi
Hideki
H
Tadakuma
Hisashi
H
Yoshida
Masasuke
M
Funatsu
Takashi
T
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D018835:Chaperonin 10 / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017510:Protein Folding; D020836:Protein Structure, Quaternary / Q000502:physiology; D013997:Time Factors
D018835:Chaperonin 10; D018834:Chaperonin 60; D010710:Phosphates; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases