Mutation Ala2-->Ser destabilizes intersubunit interactions in the molecular chaperone GroEL

Area of Science:

• Molecular biology
• Biochemistry
• Protein dynamics

Background:

• The molecular chaperone GroEL plays a critical role in protein folding.
• Understanding the allosteric regulation of GroEL's ATPase activity is crucial for its function.
• The Monod-Wyman-Changeux (MWC) model describes cooperative binding and conformational changes in proteins.

Purpose of the Study:

• To investigate the impact of the Ala2-->Ser mutation on GroEL's ATPase activity and oligomeric stability.
• To determine the effect of adenine nucleotides on the wild-type and mutant GroEL structure.
• To explore the role of GroES and rhodanese in modulating these effects.

Main Methods:

• Site-directed mutagenesis to create the Ala2-->Ser mutant of GroEL.
• Enzyme kinetics assays to measure ATP hydrolysis rates and cooperativity (Hill coefficient).
• Biophysical techniques to assess oligomeric structure stability in the presence of ligands.

Main Results:

• The Ala2-->Ser mutation significantly increased positive co-operativity in ATP hydrolysis (Hill coefficient increased from 2.36 to 3.19).
• This mutation destabilized the oligomeric structure of GroEL.
• Adenine nucleotides exhibited a pronounced destabilizing effect on the mutant GroEL.
• GroES and rhodanese binding abolished the destabilizing effect of adenine nucleotides for both wild-type and mutant GroEL.

Conclusions:

• The Ala2-->Ser mutation alters GroEL's allosteric regulation, enhancing cooperativity and reducing structural stability.
• Adenine nucleotides play a key role in modulating GroEL's stability, with this effect being mutation-dependent.
• Ligand binding (GroES, rhodanese) stabilizes GroEL against nucleotide-induced destabilization, supporting the MWC model interpretation.