GroEL Mediates Protein Folding with a Two Successive Timer Mechanism

Abstract

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.

Single-molecule Analyses of the Dynamics of Heat Shock Protein 104 (Hsp104) and Protein Aggregates

“…Total Internal Reflection Fluorescence (TIRF) MicroscopyThe experimental procedure described previously was performed (28,29), with some modifications. A flow cell, shown schematically in Fig.…”

“…Therefore, we applied single-molecule fluorescence imaging to visualize the individual dynamics between Hsp104 and aggregates to obtain information that cannot be gained from ensemble-averaged experiments. We extended our previous single-molecule experiments on the E. coli chaperonin GroEL-GroES dynamics (28,29) to the Hsp104 system and visualized the association/dissociation events of Hsp104 on the luciferase aggregates immobilized on a glass surface by TIRF microscopy (Fig. 5A).…”

“…Conventionally, TIRF illumination is used to visualize single-molecule fluorescence (e.g., Refs. 28,29). However, because TIRF microscopy only illuminates immobilized fluorescent molecules at ϳ150 nm from the surface, reliable quantitative observations of molecules larger than ϳ150 nm, which are common in protein aggregates, are not possible.…”

Multiple chaperonins in bacteria—novel functions and non-canonical behaviors

“…The intermediate domain spans two regions of the polypeptide and connects the equatorial and apical domains in sequence and structure (Saibil et al 1993;Ma et al 2000). The conformational changes resulting from ATP binding and hydrolysis at the equatorial domain are transmitted to the apical domain via this region (Hayer-Hartl et al 1995;Weissman et al 1995;Ueno et al 2004). ATP binding at the equatorial domain induces a rotation along the hinge region near the apical domain, whereby the apical domain is twisted up releasing the substrate into the cavity and exposing the hydrophobic patches for GroES to bind (Fig.…”

“…GroEL function is known to be mediated by an interplay between its interaction with and encapsulation of the substrate proteins, which are accompanied by conformational changes induced by ATP binding, hydrolysis, and GroES binding (Hayer-Hartl et al 1995;Weissman et al 1995;Ueno et al 2004). Current understanding about GroEL-GroES-mediated protein folding suggests two mechanisms: the so called cis and trans mechanisms that differ in the cavity of the GroEL where the substrate and the co-chaperonin bind.…”