A Handle-Free, All-Protein-Based Optical Tweezers Method to Probe Protein Folding-Unfolding Dynamics.

Area of Science:

• Biophysics
• Biochemistry
• Molecular Biology

Background:

• Optical tweezers (OT) are crucial for single-molecule force spectroscopy, particularly for studying protein folding-unfolding dynamics.
• Current OT methods often require attaching double-stranded DNA (dsDNA) handles to proteins, which can be inefficient and limit experimental throughput.

Purpose of the Study:

• To develop a novel, handle-free method for investigating protein folding-unfolding dynamics using optical tweezers.
• To overcome the low-yield bottleneck associated with dsDNA handle attachment in OT experiments.

Main Methods:

• Employed a handle-free, all-protein approach utilizing disordered elastin-like polypeptides (ELPs) as molecular linkers.
• Utilized the mechanically stable cohesin-dockerin (Coh-Doc) pair as a prey-bait system for efficient capture and stretching of individual protein molecules.
• Validated the method using model proteins NuG2 and RTX-v.

Main Results:

• The novel all-protein method successfully enabled the capture and stretching of individual protein molecules.
• Experimental results obtained using the ELP-Coh-Doc system were comparable to those achieved with traditional dsDNA handles.
• Demonstrated the efficiency and robustness of the handle-free approach.

Conclusions:

• Introduced a streamlined and efficient handle-free optical tweezers method for single-molecule protein dynamics studies.
• This approach expands the available techniques for single-molecule force spectroscopy, offering an alternative to dsDNA-based methods.
• Facilitates more accessible and high-throughput investigation of protein folding-unfolding mechanisms.