Hyperstable De Novo Protein with a Dimeric Bisecting Topology

Area of Science:

• Protein engineering
• Supramolecular chemistry
• Nanotechnology

Background:

• Protein nanobuilding blocks (PN-Blocks) are assembled from dimeric de novo proteins like WA20.
• WA20 has been used to create self-assembling nanostructures such as polyhedra and chain-type complexes.

Purpose of the Study:

• To stabilize the WA20 protein through targeted mutations.
• To enhance the thermal stability of the protein for improved nanostructure applications.

Main Methods:

• Designed mutations to stabilize helices and the hydrophobic core of WA20.
• Determined the midpoint denaturation temperature (Tm) of redesigned variants.
• Obtained crystal structure of the stabilized variant, Super WA20 (SUWA).
• Performed molecular dynamics simulations to analyze structural stability.

Main Results:

• The stabilized variant, Super WA20 (SUWA), showed a significantly higher Tm (122 °C) compared to WA20 (75 °C).
• Crystal structure confirmed SUWA as an intermolecularly folded dimer with a bisecting U topology.
• Molecular dynamics simulations indicated the redesigned hydrophobic core suppresses helix deformation, enhancing stability.

Conclusions:

• The redesigned hydrophobic core is critical for stabilizing the hyperstable SUWA protein.
• SUWA is a promising hyperstable de novo protein for use as nanoscale pillars in self-assembling nanoarchitectures.