AVS 45th International Symposium
    Biomaterial Interfaces Group Tuesday Sessions
       Session BI+AS+MM+NS+SS-TuA

Paper BI+AS+MM+NS+SS-TuA8
Unbinding Force of NTA-M@super 2+@--Histidine Complexes. The His-Tag Immobilization Force

Tuesday, November 3, 1998, 4:20 pm, Room 326

Session: Nanoscale to Mesocale Biomaterial Structures
Presenter: J.G. Forbes, University of Maryland, College Park
Authors: J.G. Forbes, University of Maryland, College Park
P. Yim, University of Maryland, College Park
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A sequence of six or more histidines will bind tightly to a Cu, Ni, or Co complex. The compound typically used to immobilized the metal is N-(5-amino-1-carboxypentyl)iminodiacetic acid (NTA). Most proteins will not bind to the complex unless there is a sequence of histidines, which is readily added using recombinant DNA techniques. The histidine tag may be removed from the metal complex with a high concentration of imidazole or by protonating the histidines at a pH below 6. We have studied the the unbinding strength of this interaction with the atomic force microscope (AFM). To perform this measurement, we have functionalized silicon nitride AFM tips with NTA-M@super 2+@. A glass slide was coated with recombinant DNAse I with a his-tag on the C-terminus. Unbinding force measurements were made in phosphate buffered saline (PBS) to reduce electrostatic interactions. We find that the unbinding force for the NTA-M@super 2+@/His-tag interaction to be ca. 85~pN for each of the metal complexes. Interestingly, 0.5~M imidazole does not remove the interaction, but only changes the distribution of the measured forces. This is a result of the non-equilibrium condition of the tip being forced into the protein coated surface. The interaction is almost completely removed by lowering the pH to 5.0 where the histidines are protonated and can no longer coordinate with the nickel. The remaining interaction forces are due to the histidines which are exposed when the tip presses into the surface. These results provide a quantitative measurement of mechanical strength of binding of proteins to surfaces functionalized with NTA-M@super 2+@.