Understanding domain swapping in the c-Src SH3 domain through hinge-loop mutagenesis.

Area of Science:

• Biophysics
• Structural Biology
• Protein Folding

Background:

• The c-Src SH3 domain is a well-characterized model protein.
• It exhibits noncanonical folding, forming 3D domain-swapped oligomers and amyloid fibrils.
• Understanding residues involved in unfolding and hinge loop dynamics is crucial for studying 3D domain swapping.

Purpose of the Study:

• To investigate the role of specific residues in the unfolding and 3D domain swapping of the c-Src SH3 domain.
• To construct chimeric proteins by interchanging RT and n-Src loops between c-Src SH3 and Abl SH3 domains.
• To analyze the impact of these loop interchanges on protein stability and oligomer formation.

Main Methods:

• Construction of chimeric SH3 proteins by site-directed mutagenesis.
• Interchange of RT and n-Src loop residues between c-Src SH3 and Abl SH3 domains.
• Biophysical characterization including stability assays and structural analysis of domain-swapped forms.

Main Results:

• Chimeric c-Src SH3 domain with Abl SH3 loops showed minor stability changes but retained the ability to form domain-swapped dimers.
• Interchanging one or two loops in Abl SH3 domain significantly reduced stability but did not promote 3D domain-swapped oligomer formation.
• The hinge loop composition appears to influence structural element interchange but is not the sole determinant of intertwined dimer formation.

Conclusions:

• The study elucidates the role of RT and n-Src loops in c-Src SH3 domain folding and 3D domain swapping.
• While hinge loop composition is important, other factors also contribute to the formation of 3D domain-swapped oligomers.
• Findings provide insights into the mechanisms of alternative protein folding and amyloid formation.