Several molecular biology techniques nowadays employed in the purification and immobilization of recombinant proteins are based on the formation of a coordination bond between a Ni(2+)-NTA group present on a chromatographic matrix and a stretch of six consecutive histidines (6XHis-tag) appended to the primary sequence of the protein (1). The stability of the anchoring of the His-tagged proteins on Ni(2+)-NTA functionalized matrices is challenged by the frictional force exerted on them by the flow. Such interplay between external forces and chemical processes can now be studied at the single-molecule level, thanks to the recent development in nanoscale manipulation techniques. Force spectroscopy studies performed in the past by different groups on the Ni(2+)-NTA-(His)6 bond gave rise to highly variable results (2, 3, 4). Probably, these differences derived from the different experimental setups used. We thought to overcome this problem by inserting the Ni(2+)-NTA group into a polymer with known mechanical properties, in order to have an internal control on the value of the experimentally determined force. To this aim, we thought to use ds-DNA, whose mechanical behavior has been thoroughly investigated by different authors (5, 6). We constructed a DNA molecule presenting a Ni(2+)-NTA group at one end and a thiol group at the other end. This latter group allows the attachment of the molecule to a gold-coated SFM tip via a thiol-gold bond. Force spectroscopy experiments were performed bringing the functionalized tip into proximity to a gold substrate functionalized with a CG6H6 peptide. Whenever the 6XHis-tag of the peptide formed a chelate with a Ni(2+)-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 constituted by 3 phases: (a) entropic stretching of the DNA linker; (b) overstretching transition of the linker, generating a plateau whose length must be equal to 70% of the length of the employed DNA; (c) detachment of the probe, which corresponds to the breaking of the coordination bond. In the preliminary experiments performed, the mean overstretching force resulted to be 50 pN, a value comparable to what found by other groups, while the mean length of the overstretching transition resulted 280 nm, in accordance to what expected for the DNA molecule we employed. The mean breaking force or the Ni(2+)-NTA bond resulted to be 172 pN. This value is comparable to what found by the group of Hinterdorfer et al. (3). (1) Hochuli E., H. Nobeli and A. Schacher (1987) J. Chromatogr., 411: 177-184. (2) Conti M., G. Falini and B. Samori (2000) Angew. Chem. Int. Ed., 39 (1): 215-218, (3) Kienberger F., 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. (4) Schmitt L., M. Ludwig, H. E. Gaub and R. Tampe (2000) Biophys J., 78 (6): 3275-3285. (5) Smith, S. B., Y. Cui and C. Bustamante (1996) Science. 271 (5250): 795-799. (6) Rief, M., H. Clause-Schaumann and H. E. Gaub (1999) Nature Struct. Biol. 6 (4): 346-349.

Force Spectroscopy Study of the Coordination Bond Between a Histidine Tag and the Nickel-Nitrilotriacetate Group (Ni2+-NTA).

BERGIA, ANNA;ZUCCHERI, GIAMPAOLO;SAMORI', BRUNO
2004

Abstract

Several molecular biology techniques nowadays employed in the purification and immobilization of recombinant proteins are based on the formation of a coordination bond between a Ni(2+)-NTA group present on a chromatographic matrix and a stretch of six consecutive histidines (6XHis-tag) appended to the primary sequence of the protein (1). The stability of the anchoring of the His-tagged proteins on Ni(2+)-NTA functionalized matrices is challenged by the frictional force exerted on them by the flow. Such interplay between external forces and chemical processes can now be studied at the single-molecule level, thanks to the recent development in nanoscale manipulation techniques. Force spectroscopy studies performed in the past by different groups on the Ni(2+)-NTA-(His)6 bond gave rise to highly variable results (2, 3, 4). Probably, these differences derived from the different experimental setups used. We thought to overcome this problem by inserting the Ni(2+)-NTA group into a polymer with known mechanical properties, in order to have an internal control on the value of the experimentally determined force. To this aim, we thought to use ds-DNA, whose mechanical behavior has been thoroughly investigated by different authors (5, 6). We constructed a DNA molecule presenting a Ni(2+)-NTA group at one end and a thiol group at the other end. This latter group allows the attachment of the molecule to a gold-coated SFM tip via a thiol-gold bond. Force spectroscopy experiments were performed bringing the functionalized tip into proximity to a gold substrate functionalized with a CG6H6 peptide. Whenever the 6XHis-tag of the peptide formed a chelate with a Ni(2+)-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 constituted by 3 phases: (a) entropic stretching of the DNA linker; (b) overstretching transition of the linker, generating a plateau whose length must be equal to 70% of the length of the employed DNA; (c) detachment of the probe, which corresponds to the breaking of the coordination bond. In the preliminary experiments performed, the mean overstretching force resulted to be 50 pN, a value comparable to what found by other groups, while the mean length of the overstretching transition resulted 280 nm, in accordance to what expected for the DNA molecule we employed. The mean breaking force or the Ni(2+)-NTA bond resulted to be 172 pN. This value is comparable to what found by the group of Hinterdorfer et al. (3). (1) Hochuli E., H. Nobeli and A. Schacher (1987) J. Chromatogr., 411: 177-184. (2) Conti M., G. Falini and B. Samori (2000) Angew. Chem. Int. Ed., 39 (1): 215-218, (3) Kienberger F., 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. (4) Schmitt L., M. Ludwig, H. E. Gaub and R. Tampe (2000) Biophys J., 78 (6): 3275-3285. (5) Smith, S. B., Y. Cui and C. Bustamante (1996) Science. 271 (5250): 795-799. (6) Rief, M., H. Clause-Schaumann and H. E. Gaub (1999) Nature Struct. Biol. 6 (4): 346-349.
Infmeeting: Convegno Nazionale per la Ricerca Interdisciplinare in Fisica della Materia
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Bergia A.; Zuccheri G.; Capobianco M.; Samorì B.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/13632
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