Force-dependent switch in protein unfolding pathways and transition-state movements

Area of Science:

• Biophysics
• Protein Folding Dynamics
• Single-Molecule Biophysics

Background:

• Single-domain proteins are known to fold and unfold via parallel pathways.
• Experimental demonstration of these parallel pathways has been challenging.
• Unfolding rate versus force data often shows upward curvature, suggesting pathway complexity.

Purpose of the Study:

• To provide a unified theory for protein unfolding pathway switching.
• To explain the observed upward curvature in unfolding rate as a function of perturbation.
• To investigate the structural basis for pathway switching and transition-state ensemble shifts.

Main Methods:

• Single-molecule pulling experiments on src SH3 domain.
• Development of a unified theoretical framework for protein folding landscapes.
• Molecular simulations to visualize pathway switching and transition-state ensemble dynamics.
• Analysis of protein mutants to assess landscape malleability.

Main Results:

• A unified theory demonstrates that upward curvature in unfolding rate implies a multidimensional energy landscape.
• Molecular simulations reveal structural basis for pathway switching and transition-state ensemble shifts in src SH3 domain.
• A single-point mutation altered the force at which upward curvature appears, showing landscape malleability.
• The theory successfully explains transition-state ensemble movements in other proteins (I27, monellin) under varying denaturant concentrations.

Conclusions:

• Protein unfolding pathways can switch under applied force or denaturant concentration.
• The energy landscape of protein folding is strongly multidimensional.
• Protein folding landscapes are malleable and can be tuned by mutations.
• Pathway switching is a general phenomenon predictable by the developed theory.