Tethering Complex Proteins and Protein Complexes for Optical Tweezers Experiments.

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Optical tweezers experiments require tethering proteins to force probes using DNA handles.
• Current methods often rely on thiol chemistry to link DNA to protein cysteines.
• Thiol chemistry is limited for proteins with essential or multiple cysteines at unwanted positions.

Purpose of the Study:

• To develop alternative, genetically encoded methods for attaching DNA handles to proteins.
• To overcome limitations of thiol chemistry in protein tethering for optical tweezers.
• To enable mechanical dissection of a wider range of proteins and complexes.

Main Methods:

• Utilized genetically encoded tag sequences within the target protein.
• Employed enzymatic systems: Sfp and BirA, and the SpyTag-SpyCatcher system.
• Described generation of DNA handles and creation of DNA-protein chimeras.

Main Results:

• Successfully demonstrated robust DNA handle attachment for diverse and complex proteins.
• Applied methods to proteins that are difficult to produce or purify.
• Extended application to protein complexes, such as the ribosome.

Conclusions:

• Genetically encoded tag systems provide versatile alternatives to thiol chemistry for protein tethering.
• These methods expand the scope of proteins amenable to optical tweezers analysis.
• The described approaches are valuable for studying protein mechanics when chemical methods fail.