A new atomic force microscope force ramp technique using digital force feedback control reveals mechanically weak...

Area of Science:

• Biophysics
• Materials Science
• Biotechnology

Background:

• Mechanical unfolding of proteins is crucial for understanding protein stability and function.
• Atomic Force Microscopy (AFM) is a powerful tool for probing single-molecule mechanics.
• Conventional AFM methods face challenges in controlling force loading rates during multi-step unfolding events.

Purpose of the Study:

• To develop a novel AFM force ramp modification with digital force feedback control.
• To accurately measure kinetic parameters of protein mechanical unfolding.
• To investigate mechanically weak protein species and folding kinetics.

Main Methods:

• Implemented software-based digital force feedback control for AFM force ramp modification.
• Detected unfolding events via sudden force drops and reduced applied force to a low set point.
• Applied the technique to determine unfolding kinetics of a pentamer of I27 titin domains.

Main Results:

• Achieved constant force loading rates irrespective of protein linker length or unfolded domain number.
• Obtained kinetic parameters: distance to transition state (x(u)) of 0.28 nm and unfolding rate constant at zero force (α(0)) of 1.02 × 10(-3) s(-1).
• Revealed unfolding data at low forces and detected a mechanically weak structure formed during force quench.

Conclusions:

• The developed AFM technique precisely controls protein unfolding and provides accurate kinetic parameters.
• This method enhances the study of protein mechanical unfolding, especially at low forces.
• The technique facilitates the investigation of previously hidden, mechanically weak protein species and folding pathways.