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 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.
Schmitt
L
L
Institut für Physiologische Chemie, Philipps-Universität Marburg, 35033 Marburg, Germany. schmittl@mailer.uni-marburg.de
Ludwig
M
M
Gaub
H E
HE
Tampé
R
R
D002614:Chelating Agents / Q000138:chemical synthesis*; D004492:Edetic Acid; D006639:Histidine; D008670:Metals; D018625:Microscopy, Atomic Force / Q000379:methods*; D009571:Nitrilotriacetic Acid; D010455:Peptides / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
D002614:Chelating Agents; D008670:Metals; D010455:Peptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D004492:Edetic Acid; D009571:Nitrilotriacetic Acid