A Metal-Chelating Microscopy Tip as a New Toolbox for Single-Molecule Experiments by Atomic Force Microscopy

• Published in: Biophysical journal 2000-06, Vol.78 (6), p.3275-3285
• Main Authors: Schmitt, Lutz, Ludwig, Markus, Gaub, Hermann E., Tampé, Robert
• Format: Article
• Language: English
• Subjects: Chelating Agents - chemical synthesis; Edetic Acid; Histidine; Metals; Microscopes; Microscopy, Atomic Force - methods; Nitrilotriacetic Acid; Peptides - chemistry; Recombinant Fusion Proteins - isolation & purification
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(00)76863-9

In recent years, the atomic force microscope (AFM) has contributed much to our understanding of the molecular forces involved in various high-affinity receptor-ligand systems. However, a universal anchor system for such measurements is still required. This would open up new possibilities for the study of biological recognition processes and for the establishment of high-throughput screening applications. One such candidate is the N-nitrilo-triacetic acid (NTA)/His-tag system, which is widely used in molecular biology to isolate and purify histidine-tagged fusion proteins. Here the histidine tag acts as a high-affinity recognition site for the NTA chelator. Accordingly, we have investigated the possibility of using this approach in single-molecule force measurements. Using a histidine-peptide as a model system, we have determined the binding force for various metal ions. At a loading rate of 0.5 μm/s, the determined forces varied from 22 ± 4 to 58 ± 5 pN. Most importantly, no interaction was detected for Ca 2+ and Mg 2+ up to concentrations of 10 mM. Furthermore, EDTA and a metal ion reloading step demonstrated the reversibility of the approach. Here the molecular interactions were turned off (EDTA) and on (metal reloading) in a switch-like fashion. Our results show that the NTA/His-tag system will expand the “molecular toolboxes” with which receptor-ligand systems can be investigated at the single-molecule level.