A number of extensively used methods employed in 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. Force spectroscopy studies performed in the past by different groups on the Ni2+-NTA-(His)6 bond gave rise to very different results 1, 2, 3. In order to have an internal control on the value of the experimentally determined force, we thought to insert the Ni2+-NTA group into a polymer with known mechanical properties, such as DNA 4. A DNA molecule presenting a Ni2+-NTA group at one end and a thiol group at the other end was attached to a gold surface via a thiol-gold bond. Force spectroscopy experiments were performed bringing a gold-coated AFM tip functionalized with a CG6H6 peptide into proximity to the NTA-DNA gold substrate. Whenever the 6XHis-tag of the peptide formed a chelate with a Ni2+-NTA group appended at the end of the DNA linker, a molecular bridge was established between the tip and the substrate. The tip was subsequently retracted until the bridge broke and the resulting force/distance curve was collected. The identification of the formation of the desired coordination bond is easy since it leads to a force curve in which the overstretching transition of the DNA linker generates a plateau whose length must be equal to 70% of the length of the DNA. In the preliminary experiments performed, the mean breaking force or the Ni2+-NTA bond resulted to be 172 pN. This value is comparable to what found by the group of Hinterdorfer et al. 2. 1 M. Conti, G. Falini and B. Samori (2000) Angew. Chem. Int. Ed., 39 (1): 215-218, 2 F. Kienberger, G. Kada, H. J. Gruber, V. P. Pastushenko, C. Reiner, M. Trieb, H.-G. Knaus, H. Schindler and P. Hinterdorfer (2000) Single Mol., 1: 59-65. 3 Schmitt L., M. Ludwig, H. E. Gaub and R. Tampe (2000) Biophys J., 78 (6): 3275-3285. 4 Smith, S. B., Y. Cui and C. Bustamante (1996) Science. 271 (5250): 795-799.
Force Spectroscopy Study of the Coordination Bond Between a His-Tag and the (Ni2+-NTA) Group.
BERGIA, ANNA;ZUCCHERI, GIAMPAOLO;SAMORI', BRUNO
2004
Abstract
A number of extensively used methods employed in 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. Force spectroscopy studies performed in the past by different groups on the Ni2+-NTA-(His)6 bond gave rise to very different results 1, 2, 3. In order to have an internal control on the value of the experimentally determined force, we thought to insert the Ni2+-NTA group into a polymer with known mechanical properties, such as DNA 4. A DNA molecule presenting a Ni2+-NTA group at one end and a thiol group at the other end was attached to a gold surface via a thiol-gold bond. Force spectroscopy experiments were performed bringing a gold-coated AFM tip functionalized with a CG6H6 peptide into proximity to the NTA-DNA gold substrate. Whenever the 6XHis-tag of the peptide formed a chelate with a Ni2+-NTA group appended at the end of the DNA linker, a molecular bridge was established between the tip and the substrate. The tip was subsequently retracted until the bridge broke and the resulting force/distance curve was collected. The identification of the formation of the desired coordination bond is easy since it leads to a force curve in which the overstretching transition of the DNA linker generates a plateau whose length must be equal to 70% of the length of the DNA. In the preliminary experiments performed, the mean breaking force or the Ni2+-NTA bond resulted to be 172 pN. This value is comparable to what found by the group of Hinterdorfer et al. 2. 1 M. Conti, G. Falini and B. Samori (2000) Angew. Chem. Int. Ed., 39 (1): 215-218, 2 F. Kienberger, G. Kada, H. J. Gruber, V. P. Pastushenko, C. Reiner, M. Trieb, H.-G. Knaus, H. Schindler and P. Hinterdorfer (2000) Single Mol., 1: 59-65. 3 Schmitt L., M. Ludwig, H. E. Gaub and R. Tampe (2000) Biophys J., 78 (6): 3275-3285. 4 Smith, S. B., Y. Cui and C. Bustamante (1996) Science. 271 (5250): 795-799.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
