Single Molecule Force Spectroscopy study of the coordination bond between a histidine tag and the nickel-nitrilotriacetate group.

Nowadays several methods used for the purification of recombinant proteins are based on the formation of a coordination bond between the nickel(II)-nitrilotriacetate (NTA) group present on a chromatographic matrix and a stretch of six consecutive histidine residues (6XHis-tag) appended to the primary sequence of the protein [1]. Measuring the force anchoring the His-tagged proteins on the Ni2+-NTA functionalized matrices at the single molecule level can provide an insight in the chemical details of the bond, which could then be used to design possible improvement strategies. This approach has been already explored by different groups which measured the force of the Ni2+-NTA-(His)6 bond obtaining highly variable results [2,3,4]. Probably, these differences derived from the different experimental setups used. We faced such a problem by using an internal force gauge constituted by a dsDNA linker tethering the nitrilotriacetate (NTA) group to the surface. The mechanical properties of dsDNA have been in fact thoroughly investigated by different groups at the single molecule level [5,6]. We constructed a DNA molecule presenting a Ni2+-NTA group at one end and a thiol group, for the binding onto a gold surface, at the other end. Force-distance curves between the AFM tip, previously functionalised with a CG6H6 peptide, and the DNA bound to the surface were collected. The formation of the desired coordination bond leads to a force curve with 3 phases: (1) entropic stretching of the DNA linker; (2) overstretching transition of the linker, generating a plateau whose length is equal to 70% of that of the employed DNA; (3) detachment of the probe, which corresponds to the breaking of the coordination bond. Preliminary results show the overstretching plateau at 50 pN, a value close to the earlier reported ones, followed by the two possible rupture force profiles: either a single entropic stretching represented by classical Worm Like Chain (WLC) or a more complex profile which seems to be the combination of two WLC. This means that there are two possible scenarios in our experiments. In the first case, after the overstretch, the DNA linker (in the S-DNA form) is extended till the force reaches the value of the rupture of the Ni2+-NTA-(His)6 bond; in the second case the stretching of the S-DNA is followed by a melting process leading to the partial separation of the DNA strands. A description of the experiments and the values of the forces measured for the Ni2+-NTA-(His)6 bonds, which seems in agreement with the one measured by the Hinterdorfer group [3], will be presented. [1] Hochuli E et al. (1987) J. Chromatogr., 411: 177. [2] Conti M et al. (2000) Angew. Chem. Int. Ed., 39: 215. [3] Kienberger F. et al (2000) Single Mol., 1: 59. [4] Schmitt L. et al. (2000) Biophys J., 78: 3275. [5] Smith, S. B. et al. (1996) Science. 271: 795. [6] Rief, M. et al. (1999) Nature Struct. Biol. 6: 346.