Edge strand engineering prevents native-like aggregation in Sulfolobus solfataricus acylphosphatase

Area of Science:

• Biochemistry and structural biology
• Protein aggregation and stability
• Enzyme engineering

Background:

• Beta-proteins risk aggregation due to edge-to-edge beta-sheet interactions.
• Acylphosphatase from Sulfolobus solfataricus (Sso AcP) aggregates via N-terminal segment interaction with beta-strand B4.
• Engineered Sso AcP mutants (V84D, Y86E, V84P) were designed to reduce aggregation.

Purpose of the Study:

• To elucidate the structural basis for reduced aggregation and lower stability in Sso AcP mutants.
• To understand the mechanism by which mutations in beta-strand B4 affect aggregation propensity.
• To provide a foundation for designing proteins resistant to aggregation.

Main Methods:

• X-ray crystallography to determine the structures of Sso AcP variants (V84P, V84D, Y86E).
• Analysis of structural changes in beta-strand B4 and surrounding regions.
• Thermodynamic and kinetic experiments to assess protein stability and aggregation mechanisms.

Main Results:

• V84D and Y86E mutations introduce negative charge at the B4 edge, hindering aggregation.
• V84P mutation results in a less conformationally regular B4, reducing aggregation.
• Mutant aggregation is non-native-like and independent of the N-terminal segment interaction.

Conclusions:

• Specific mutations in Sso AcP effectively impair native-like aggregation by modifying beta-strand B4.
• The structural insights gained can guide the rational design of aggregation-resistant proteins.
• Reduced stability is a trade-off for enhanced aggregation resistance in these engineered variants.