Tracking unfolding and refolding reactions of single proteins using atomic force microscopy methods

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Single-molecule manipulation techniques, particularly atomic force microscopy (AFM), have become crucial for understanding biomolecular structure and function.
• AFM provides unparalleled resolution for observing molecular processes in real-time.

Purpose of the Study:

• To detail the application of single-molecule AFM for tracking protein unfolding and refolding pathways.
• To investigate the impact of osmolytes and chaperones on protein stability and folding dynamics.
• To describe AFM operational principles and experimental protocols.

Main Methods:

• Utilizing single-molecule atomic force microscopy (AFM) to monitor protein behavior.
• Employing AFM pulling techniques, including length clamp and force-clamp modes.
• Developing protocols for polyprotein construction, sample preparation, and cantilever calibration.

Main Results:

• Demonstrated the capacity of AFM to track dynamic protein processes like unfolding and refolding.
• Illustrated the influence of external factors (osmolytes, chaperones) on protein stability.
• Provided a comprehensive guide to AFM experimental setup and data analysis.

Conclusions:

• Single-molecule AFM is a powerful tool for elucidating complex protein dynamics and stability mechanisms.
• The described methods and protocols facilitate advanced research in protein science using AFM.
• AFM offers fundamental insights into biomolecular function and interactions.