L. Schmitt, L. Schmitt, M. Ludwig
Jun 1, 2000
Citations
3
Influential Citations
153
Citations
Quality indicators
Journal
Biophysical journal
Abstract
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 microm/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.