Thermally denatured state determines refolding in lipase: mutational analysis

Area of Science:

• Biochemistry
• Protein Engineering
• Molecular Biology

Background:

• Protein aggregation during thermal denaturation limits thermodynamic characterization and thermostability studies.
• Understanding mutations that prevent aggregation offers insights into protein folding pathways.

Purpose of the Study:

• To identify mutations that prevent irreversible thermal aggregation in Bacillus subtilis lipase.
• To investigate the mechanisms by which these mutations enhance protein thermotolerance.

Main Methods:

• Directed evolution and site-directed mutagenesis to generate lipase variants.
• Thermal denaturation assays and residual activity measurements.
• Characterization of aggregation-prone intermediates using bis-ANS binding and solubility assays.

Main Results:

• A single mutation, M137P, completely prevented thermal aggregation in Bacillus subtilis lipase.
• Mutants M137P, M134E, and S163P partially or completely prevented aggregation-prone intermediate formation.
• Mutations showed marginal changes in free energy of unfolding but significant shifts in thermostability profiles.
• Charge-mediated interactions in M134E were implicated in preventing aggregation.

Conclusions:

• Specific mutations can prevent thermal aggregation by inhibiting the formation of aggregation-prone intermediates.
• These mutations represent a novel strategy for enhancing protein thermotolerance.
• The identified mutations influence the denatured ensemble, not intrinsic protein stability.