Single-molecule observation of protein–protein interactions in the chaperonin system

Abstract

We have analyzed the dynamics of the chaperonin (GroEL)-cochaperonin (GroES) interaction at the single-molecule level. In the presence of ATP and non-native protein, binding of GroES to the immobilized GroEL occurred at a rate that is consistent with bulk kinetics measurements. However, the release of GroES from GroEL occurred after a lag period ( approximately 3 s) that was not recognized in earlier bulk-phase studies. This observation suggests a new kinetic intermediate in the GroEL-GroES reaction pathway.

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.…”

Single-molecule spectroscopy of protein folding in a chaperonin cage

“…Even the autonomous folding of chaperone substrate proteins has been difficult to investigate because of their strong aggregation tendency (10). Contributions from confinement and crowding have been addressed in numerous studies using molecular simulations and theory (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), but many of these concepts have eluded experimental examination.Here, we take a step towards closing this gap by investigating the GroEL/GroES chaperonin (1-3, 9) with single-molecule fluorescence spectroscopy (21-24), a method that is starting to provide previously inaccessible information on chaperonemediated protein folding (25)(26)(27)(28)(29)(30). GroEL/GroES is a remarkable molecular machine that binds nonnative proteins and allows them to fold within a cavity formed by the heptameric rings of GroEL and GroES.…”

“…Here, we take a step towards closing this gap by investigating the GroEL/GroES chaperonin (1-3, 9) with single-molecule fluorescence spectroscopy (21-24), a method that is starting to provide previously inaccessible information on chaperonemediated protein folding (25)(26)(27)(28)(29)(30). GroEL/GroES is a remarkable molecular machine that binds nonnative proteins and allows them to fold within a cavity formed by the heptameric rings of GroEL and GroES.…”