Nanometre localization of single ReAsH molecules

Area of Science:

• Biophysics
• Molecular Biology
• Nanotechnology

Background:

• ReAsH is a red-fluorescent dye that selectively binds to tetracysteine motifs in proteins.
• Calmodulin is a crucial calcium-binding protein involved in cellular signaling.
• Precise tracking of single molecules is essential for studying biomolecular mechanisms.

Purpose of the Study:

• To develop and validate a high-precision method for tracking single ReAsH-labeled calmodulin molecules.
• To characterize the photostability and imaging parameters of ReAsH.
• To demonstrate the capability of observing nanoscale translational motion of biomolecules.

Main Methods:

• Immobilization of single calmodulin molecules with an inserted tetracysteine motif on a glass surface.
• Total internal reflection fluorescence microscopy (TIRFm) for single-molecule imaging.
• Precise localization of the dye's position using photon distribution analysis.
• Characterization of ReAsH photostability and the effect of 2-mercaptoethanesulphonic acid.
• Nanoscale manipulation of ReAsH molecules using a nanometric stage.

Main Results:

• Single ReAsH molecules were localized with 5 nm precision in 0.5 s.
• ReAsH exhibited photostability allowing observations from seconds to over a minute.
• 2-mercaptoethanesulphonic acid enhanced photon collection by a factor of two.
• Translational motion in 25-40 nm steps was clearly observed, demonstrating the technique's capability.

Conclusions:

• High-precision single-molecule tracking of ReAsH-labeled proteins is feasible.
• The developed method is suitable for observing nanoscale movements relevant to biomolecular motors.
• ReAsH imaging can be optimized for enhanced signal and observation duration.