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The NTA–His 6 bond is strong enough for AFM single‐molecular recognition studies
Author(s) -
Verbelen Claire,
Gruber Hermann J.,
Dufrêne Yves F.
Publication year - 2007
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.833
Subject(s) - nitrilotriacetic acid , force spectroscopy , chemistry , atomic force microscopy , dissociation (chemistry) , moiety , context (archaeology) , crystallography , nanotechnology , molecule , stereochemistry , chelation , organic chemistry , materials science , paleontology , biology
Abstract There is a need in current atomic force microscopy (AFM) molecular recognition studies for generic methods for the stable, functional attachment of proteins on tips and solid supports. In the last few years, the site‐directed nitrilotriacetic acid (NTA)–polyhistidine (His n ) system has been increasingly used towards this goal. Yet, a crucial question in this context is whether the NTA–His n bond is sufficiently strong for ensuring stable protein immobilization during force spectroscopy measurements. Here, we measured the forces between AFM tips modified with NTA‐terminated alkanethiols and solid supports functionalized with His 6 ‐Gly‐Cys peptides in the presence of Ni 2+ . The force histogram obtained at a loading rate of 6600 pN s −1 showed three maxima at rupture forces of 153 ± 57 pN, 316 ± 50 pN and 468 ± 44 pN, that we attribute primarily to monovalent and multivalent interactions between a single His 6 moiety and one, two and three NTA groups, respectively. The measured forces are well above the 50–100 pN unbinding forces typically observed by AFM for receptor–ligand pairs. The plot of adhesion force versus log (loading rate) revealed a linear regime, from which we deduced a kinetic off‐rate constant of dissociation, k off ∼0.07 s −1 . This value is in the range of that estimated for the multivalent interaction involving two NTA, using fluorescence measurements, and may account for an increased binding stability of the NTA–His 6 bond. We conclude that the NTA–His 6 system is a powerful, well‐suited platform for the stable, oriented immobilization of proteins in AFM single‐molecule studies. Copyright © 2007 John Wiley & Sons, Ltd.