A simple two-state protein unfolds mechanically via multiple heterogeneous pathways at single-molecule resolution

Area of Science:

• Biophysics
• Molecular Biology
• Computational Chemistry

Background:

• Understanding protein folding mechanisms is crucial for molecular biology and drug development.
• Experimental methods struggle to resolve the complexity of protein folding pathways.
• Two-state protein folding is a simplified model that may mask underlying complexity.

Purpose of the Study:

• To investigate the mechanical unfolding of cold shock protein B (Csp) using single-molecule force spectroscopy.
• To reveal the microscopic pathways and mechanisms of protein folding.
• To validate theoretical and computational simulation predictions of protein folding.

Main Methods:

• Single-molecule force spectroscopy was used to study the mechanical unfolding of Csp.
• Steered molecular dynamics simulations were employed to model the unfolding process.
• Experimental and simulation data were compared to match unfolding patterns with structural events.

Main Results:

• Csp unfolding under moderate force exhibited high heterogeneity.
• Unfolding trajectories included single sweeps and multiple, long-lived mechanical intermediates.
• The order of intermediate appearance varied across different unfolding events.
• Steered molecular dynamics simulations accurately reproduced the experimental observations.

Conclusions:

• Two-state protein folding can exhibit significant nanoscale complexity.
• Combined experimental and computational methods offer unique insights into protein unfolding mechanisms.
• This approach can dissect the mechanical unfolding of proteins with complex folding behaviors.