Multivalent binding of nonnative substrate proteins by the chaperonin GroEL.

Area of Science:

• Molecular Chaperones
• Protein Folding
• Biochemistry

Background:

• The chaperonin GroEL facilitates protein folding by binding nonnative substrates.
• GroEL's mechanism involves ATP binding and cochaperonin GroES interaction.
• The number of GroEL apical domains involved in substrate binding remains unclear.

Purpose of the Study:

• To investigate the stoichiometry of nonnative protein binding to GroEL apical domains.
• To determine the minimum number and arrangement of binding-proficient domains required for substrate interaction.

Main Methods:

• Utilized engineered GroEL rings with combinations of wild-type and binding-defective mutant apical domains.
• Assessed binary complex formation with stringent (malate dehydrogenase, Rubisco) and less stringent (rhodanese) substrate proteins.
• Employed oxidative cross-linking to physically confirm multivalent binding of Rubisco.

Main Results:

• A minimum of three consecutive wild-type apical domains were necessary for efficient binding of stringent substrates.
• Less stringent substrate rhodanese required only two binding domains, irrespective of their arrangement.
• Direct evidence of multivalent Rubisco binding to GroEL was obtained via cross-linking.

Conclusions:

• GroEL substrate binding is multivalent, with stringent proteins requiring multiple interaction sites.
• The number of required binding domains correlates with substrate stringency.
• This study elucidates the quantitative aspects of substrate recognition by the GroEL chaperonin system.