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Biophys J. 2000 Jun;78(6):3275-85.

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

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1
Institut für Physiologische Chemie, Philipps-Universität Marburg, 35033 Marburg, Germany. schmittl@mailer.uni-marburg.de

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.

PMID:
10828003
PMCID:
PMC1300908
DOI:
10.1016/S0006-3495(00)76863-9
[Indexed for MEDLINE]
Free PMC Article
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