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

被引:140
作者
Schmitt, L [1 ]
Ludwig, M
Gaub, HE
Tampé, R
机构
[1] Univ Marburg, Inst Physiol Chem, D-35033 Marburg, Germany
[2] Univ Munich, Lehrstuhl Angew Phys, D-80799 Munich, Germany
关键词
D O I
10.1016/S0006-3495(00)76863-9
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
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 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 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.
引用
收藏
页码:3275 / 3285
页数:11
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