Requirement for binding multiple ATPs to convert a GroEL ring to the folding-active state

Area of Science:

• Molecular biology
• Protein folding
• Biochemistry

Background:

• The GroEL chaperonin system facilitates protein folding through a cycle of substrate binding and release.
• This cycle is regulated by ATP binding and hydrolysis, coupled with the binding and release of the co-chaperonin GroES.
• The precise number of ATP-bound subunits required to trigger key events remains unclear.

Purpose of the Study:

• To determine the minimum number of ATP-bound subunits in a GroEL ring necessary for GroES binding and subsequent substrate protein release and folding.
• To investigate the quantitative relationship between ATP binding and the mechanical events driving protein refolding.

Main Methods:

• Construction of GroEL rings with varying ratios of wild-type and nucleotide-binding-deficient mutant subunits.
• Utilizing a specific inhibitor to block ATP binding and turnover in mutant subunits.
• Monitoring GroES binding, substrate protein release, and refolding efficiency in response to different subunit compositions.

Main Results:

• A minimum of two wild-type subunits are required for efficient GroES binding to the GroEL ring.
• At least four wild-type subunits are necessary to trigger substrate polypeptide release and initiate folding.
• Maximal refolding efficiency is achieved when all seven subunits are wild-type, suggesting a cumulative effect.

Conclusions:

• The GroEL chaperonin requires a threshold of ATP-bound subunits to initiate distinct functional steps.
• Substrate release and folding are driven by a cooperative mechanism involving multiple ATP-bound subunits, likely through a conformational "power stroke."
• Understanding subunit cooperativity is crucial for elucidating the mechanism of ATP-dependent chaperones.