The folding transition state between SH3 domains is conformationally restricted and evolutionarily conserved

Area of Science:

• Protein engineering
• Biochemistry
• Structural biology

Background:

• The alpha-spectrin SH3 domain is a small protein module involved in protein-protein interactions.
• Understanding protein folding mechanisms is crucial for comprehending protein function and dysfunction.

Purpose of the Study:

• To investigate the folding transition state of the alpha-spectrin SH3 domain under varying pH conditions.
• To compare the folding mechanism with homologous SH3 domains, such as the Src SH3 domain.
• To elucidate the role of polypeptide topology and chain connectivity in protein folding.

Main Methods:

• Protein engineering analysis of the alpha-spectrin SH3 domain.
• Stability measurements at different pH values (7.0, 3.5, and 2.5).
• Brønsted plot analysis to determine folding subdomains.
• Comparative analysis with the Src SH3 domain.

Main Results:

• The folding transition state is structured around the distal loop beta-hairpin and 310-helix, showing minimal conformational variability.
• This transition state structure appears conserved across homologous SH3 domains, despite sequence differences.
• Brønsted plot analysis identified two distinct folding subdomains within the alpha-spectrin SH3 domain.
• Polypeptide topology and chain connectivity significantly influence the folding pathway.

Conclusions:

• The alpha-spectrin SH3 domain exhibits a highly conserved and conformationally stable folding transition state.
• Protein folding in this family is strongly dictated by topological constraints and how the polypeptide chain is connected.
• These findings contribute to a deeper understanding of protein folding principles and domain conservation.