Circularization changes the folding transition state of the src SH3 domain

Area of Science:

• Protein folding dynamics
• Biophysics
• Structural biology

Background:

• Protein native state topology influences folding pathways.
• The src SH3 domain is a model system for studying protein folding.
• Transition state ensembles are critical for understanding folding mechanisms.

Purpose of the Study:

• To experimentally investigate the robustness of the src SH3 domain's folding transition state to topological changes.
• To determine how covalent crosslinking affects protein folding kinetics and transition state stability.
• To elucidate the relationship between topology, sequence, and the protein free energy landscape.

Main Methods:

• Disulfide crosslinking to alter protein topology.
• Kinetic analysis of point mutations in modified proteins.
• Circular dichroism and other biophysical techniques to characterize transition states.

Main Results:

• Circularization of the src SH3 domain led to topological symmetry and delocalization of structure in the transition state ensemble, suggesting a modified folding mechanism.
• Crosslinking the distal beta-hairpin accelerated folding 30-fold without altering transition state structure distribution.
• These findings indicate that the free energy landscape is sensitive to topological alterations.

Conclusions:

• Protein folding landscapes possess deep features robust to sequence variation but sensitive to topological modifications.
• Topology plays a crucial role in dictating protein folding mechanisms and kinetics.
• The src SH3 domain's folding is adaptable to topological constraints, highlighting the complex interplay of factors governing protein structure formation.