Protein folding mechanism revealed by single-molecule force spectroscopy experiments.

Area of Science:

• Biophysics
• Protein dynamics
• Mechanical spectroscopy

Background:

• Force spectroscopy is crucial for understanding protein folding and unfolding.
• Atomic force microscopy (AFM) has been extensively used for high-force measurements.
• Optical tweezers and magnetic tweezers enable low-force measurements, probing larger conformational spaces.

Purpose of the Study:

• To review force spectroscopy experiments on protein folding and unfolding.
• To compare high-force and low-force measurement results.
• To elucidate the general protein folding mechanism through low-force studies.

Main Methods:

• Atomic force microscopy (AFM) for high-force protein unfolding.
• Optical tweezers and magnetic tweezers for low-force protein folding/unfolding.
• Analysis of unfolding forces and distances to map free energy landscapes.

Main Results:

• High-force unfolding distances typically < 2 nm (AFM).
• Low-force unfolding distances range from negative values to > 6 nm.
• Transition state sizes at low force are ~4 nm for globular proteins, suggesting a molten globule intermediate.

Conclusions:

• Low-force spectroscopy offers detailed free energy landscape information.
• The ~4 nm transition state may represent a general barrier between unfolded and molten globule states.
• Further low-force studies on diverse proteins are needed to fully understand protein folding mechanisms.