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

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 measu rements is still required. This would open up new possibilities for the stu dy of biological recognition processes and for the establishment of high-th roughput screening applications. One such candidate is the N-nitrilo-triace tic acid (NTA)/His-tag system, which is widely used in molecular biology to isolate and purify histidine-tagged fusion proteins. Here the histidine ta g acts as a high-affinity recognition site for the NTA chelator. Accordingl y, we have investigated the possibility of using this approach in single-mo lecule 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 mu m/s, the determined forces varied from 22 +/- 4 to 58 +/- 5 pN. Most importantly, no interaction was detected for Ca2+ and Mg2+ up to conce ntrations of 10 mM. Furthermore, EDTA and a metal ion reloading step demons trated the reversibility of the approach. Here the molecular interactions w ere turned off (EDTA) and on (metal reloading) in a switch-like fashion. Ou r results show that the NTA/His-tag system will expand the "molecular toolb oxes" with which receptor-ligand systems can be investigated at the single- molecule level.