Changes of protein stiffness during folding detect protein folding intermediates

Area of Science:

• Biophysics
• Protein Folding Dynamics
• Single-Molecule Techniques

Background:

• Understanding protein folding is crucial for molecular biology.
• Identifying folding intermediates provides insights into protein stability and function.
• Existing methods face challenges in resolving transient states during folding.

Purpose of the Study:

• To detect and characterize folding intermediates of a simple protein.
• To investigate changes in molecular stiffness during protein folding.
• To validate experimental findings with theoretical models.

Main Methods:

• Utilized single-molecule force-quench atomic force microscopy (FQ-AFM).
• Analyzed autocorrelation of fluctuations in end-to-end length to determine molecular stiffness.
• Applied equipartition theorem and Langevin dynamics simulations for support.

Main Results:

• Detected changes in molecular stiffness indicative of folding intermediates.
• Stiffness changes were derived from the shape and peaks of autocorrelation functions.
• Experimental data align with Langevin dynamics simulations.

Conclusions:

• FQ-AFM is effective in probing protein folding intermediates.
• The study identifies an ensemble of random-coiled collapsed states in protein folding pathways.
• Results support the presence of these states in both force-quench and thermal-quench folding.