A metal-chelating microscopy tip as a new toolbox for single-molecule experiments by atomic force microscopy
A metal-chelating microscopy tip as a new toolbox for single-molecule experiments by atomic force microscopy
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DOI：
10.1016/s0006-3495(00)76863-9
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发表时间：
2000-06-01
影响因子：
3.4
通讯作者：
Tampé, R
中科院分区：
文献类型：
--
作者：
Schmitt, L;Ludwig, M;Tampé, R
近年来，原子力显微镜（AFM）为我们理解各种高亲和力受体-配体系统中的分子力做出了很大贡献。然而，仍然需要用于这种测量的通用锚系统。这将为研究生物识别过程和建立高通量筛选应用开辟新的可能性。其中一个候选系统是N-次氮基三乙酸（NTA）/His标签系统，该系统广泛用于分子生物学中分离和纯化带有组氨酸标签的融合蛋白。在这里，组氨酸标签充当NTA螯合剂的高亲和力识别位点。因此，我们研究了使用这种方法在单分子力测量的可能性。使用组氨酸肽作为模型系统，我们已经确定了各种金属离子的结合力。在0.5 μ m/s的加载速率下，测定的力从22 +/- 4到58 +/- 5 pN不等。最重要的是，没有相互作用检测到的Ca 2+和Mg 2+的浓度高达10 mM。此外，EDTA和金属离子重新加载步骤证明了该方法的可逆性。在这里，分子间的相互作用以类似开关的方式被关闭（EDTA）和打开（金属重新加载）。我们的研究结果表明，NTA/His标签系统将扩大“分子工具箱”，与受体配体系统可以在单分子水平上进行研究。
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.