A metal-chelating microscopy tip as a new toolbox for single-molecule experiments by atomic force microscopy

Area of Science:

• Biophysics
• Molecular Biology
• Nanotechnology

Background:

• Atomic force microscopy (AFM) is crucial for understanding molecular forces in receptor-ligand systems.
• A universal anchoring system is needed to advance studies of biological recognition and high-throughput screening.
• The N-nitrilo-triacetic acid (NTA)/His-tag system is widely used for protein purification and shows potential for single-molecule force measurements.

Purpose of the Study:

• To investigate the NTA/His-tag system as a universal anchor for single-molecule force measurements.
• To determine the binding forces of the NTA/His-tag system with various metal ions.
• To assess the reversibility and switchable nature of the NTA/His-tag interaction.

Main Methods:

• Utilized atomic force microscopy (AFM) for single-molecule force measurements.
• Employed a histidine-peptide as a model system.
• Tested the NTA/His-tag interaction with different metal ions, including Ca(2+) and Mg(2+).
• Investigated reversibility using EDTA and metal ion reloading.

Main Results:

• The NTA/His-tag system demonstrated switchable binding forces ranging from 22 +/- 4 to 58 +/- 5 pN at a loading rate of 0.5 µm/s.
• No interaction was detected for Ca(2+) and Mg(2+) up to 10 mM concentrations.
• The system exhibited reversible on/off switching of molecular interactions upon metal ion chelation and reloading.

Conclusions:

• The NTA/His-tag system is a viable and versatile tool for single-molecule force measurements.
• This system expands the capabilities for investigating receptor-ligand interactions at the single-molecule level.
• The switchable nature of the NTA/His-tag interaction facilitates applications in biological recognition studies and high-throughput screening.