Creating the Functional Single-Ring GroEL-GroES Chaperonin Systems via Modulating GroEL-GroES Interaction

Area of Science:

• Molecular Biology
• Protein Folding
• Evolutionary Biology

Background:

• Chaperonins like E. coli GroEL and cochaperonins like GroES are crucial for protein folding.
• The functional interaction requires a double-ring GroEL assembly with GroES, while a single-ring GroEL variant (GroEL^SR) arrests the cycle with GroES.
• Mutations in GroES at residue I25 disrupt GroEL interaction, amplified by its heptameric structure.

Purpose of the Study:

• To create GroES variants with weakened GroEL^SR interaction by incorporating I25 mutations into a multi-module GroES construct (GroES⁷).
• To investigate how the number and arrangement of mutated modules in GroES⁷ affect its interaction with GroEL^SR and its ability to facilitate protein folding.
• To provide insights into the evolution of bacterial chaperonin systems and their relation to human mitochondrial chaperonins.

Main Methods:

• Construction of GroES⁷ variants with varying numbers and positions of I25A or I25D mutations.
• Biochemical characterization of GroES⁷-GroEL^SR interactions.
• In vitro and in vivo assays to assess protein folding and chaperone function.

Main Results:

• GroES⁷ variants with two mutated modules restored GroEL^SR-mediated protein folding in vitro.
• GroES⁷ variants containing two or three mutated modules enabled GroEL^SR to perform chaperone functions in vivo under normal and heat-shock conditions.
• Three specific GroES⁷ variants demonstrated robust function with GroEL^SR in vivo.

Conclusions:

• Controlled weakening of GroEL^SR-GroES interaction using engineered GroES⁷ variants can restore chaperone function.
• The number and arrangement of mutated modules in GroES⁷ are critical for functional restoration.
• These findings offer valuable insights into the functional adaptation of bacterial chaperonin systems and their evolutionary link to human mitochondrial chaperonins (mtHsp60-mtHsp10).