Triggering protein folding within the GroEL-GroES complex.

Area of Science:

• * Molecular biology
• * Biochemistry
• * Structural biology

Background:

• * Protein folding is crucial for cellular function and often requires molecular chaperones.
• * Chaperonins, such as GroEL and GroES, assist protein folding by encapsulating substrates within a central cavity.
• * The precise mechanism of substrate encapsulation and release by the GroEL-GroES complex remains incompletely understood.

Purpose of the Study:

• * To elucidate the mechanism of substrate protein encapsulation and folding by the GroEL-GroES chaperonin system.
• * To investigate the role of allosteric states in the GroEL-GroES cycle.
• * To decouple substrate encapsulation from release and folding using a novel GroEL mutant.

Main Methods:

• * Employed a chemically modified GroEL mutant (EL43Py) designed to stall in an intermediate conformational state.
• * Analyzed the binding affinities of the mutant GroEL for both GroES and non-native substrate proteins.
• * Investigated the kinetics of ATP hydrolysis and complex disassembly in the GroEL-GroES system.

Main Results:

• * The EL43Py mutant successfully initiated substrate encapsulation but remained stalled in an intermediate allosteric state.
• * This intermediate state exhibited high affinity for both GroES and substrate proteins, preventing premature release.
• * The assembly of the functional GroEL-GroES complex involves a delayed ATP hydrolysis, linked to the disassembly of the opposing ring's complex.

Conclusions:

• * A specific intermediate allosteric state of GroEL is critical for both GroES binding and substrate protein unfolding.
• * Substrate protein encapsulation and compaction occur prior to release, facilitated by this stalled intermediate.
• * The functional cycle of the GroEL-GroES chaperonin involves a regulated delay in ATP hydrolysis and coordinated ring disassembly.