Sequential unfolding of beta helical protein by single-molecule atomic force microscopy

Area of Science:

• Biophysics
• Structural Biology
• Microbiology

Background:

• Extracellular proteins in Gram-negative bacteria often feature a parallel beta-helix fold.
• The mechanical properties of these beta-helical proteins are largely uncharacterized.
• The TpsA protein family exemplifies this fold across diverse functions.

Purpose of the Study:

• To investigate the mechanical properties of the TpsA protein family using a prototypic adhesin.
• To elucidate the unfolding pathway and identify mechanically stable regions within the beta-helix.

Main Methods:

• Single-molecule atomic force microscopy (AFM) was employed to probe protein mechanics.
• Steered molecular dynamics (SMD) simulations were utilized to complement experimental data.

Main Results:

• High forces were needed to fully unfold the FHA protein, indicating significant mechanical stability.
• Unfolding proceeded in a stepwise, hierarchical manner, with extremities unfolding before central regions.
• A conserved, mechanically resistant subdomain critical for secretion was identified within the TpsA family.

Conclusions:

• The hierarchical unfolding mechanism of the beta-helix may facilitate structural recovery after mechanical stress.
• Identified mechanical properties are likely relevant to other beta-helical proteins in prokaryotes and eukaryotes.
• Understanding these properties is crucial for protein structural integrity and function.