title: Antibody-unfolding and metastable-state binding in force spectroscopy and recognition imaging.
abstract: Force spectroscopy and recognition imaging are important techniques for characterizing and mapping molecular interactions. In both cases, an antibody is pulled away from its target in times that are much less than the normal residence time of the antibody on its target. The distribution of pulling lengths in force spectroscopy shows the development of additional peaks at high loading rates, indicating that part of the antibody frequently unfolds. This propensity to unfold is reversible, indicating that exposure to high loading rates induces a structural transition to a metastable state. Weakened interactions of the antibody in this metastable state could account for reduced specificity in recognition imaging where the loading rates are always high. The much weaker interaction between the partially unfolded antibody and target, while still specific (as shown by control experiments), results in unbinding on millisecond timescales, giving rise to rapid switching noise in the recognition images. At the lower loading rates used in force spectroscopy, we still find discrepancies between the binding kinetics determined by force spectroscopy and those determined by surface plasmon resonance-possibly a consequence of the short tethers used in recognition imaging. Recognition imaging is nonetheless a powerful tool for interpreting complex atomic force microscopy images, so long as specificity is calibrated in situ, and not inferred from equilibrium binding kinetics.
authors.affiliation: Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
authors.forename: Parminder
authors.initials: P
authors.lastname: Kaur
authors.lastname: Qiang-Fu
authors.forename: Alexander
authors.initials: A
authors.lastname: Fuhrmann
authors.forename: Robert
authors.initials: R
authors.lastname: Ros
authors.forename: Linda Obenauer
authors.initials: LO
authors.lastname: Kutner
authors.forename: Lumelle A
authors.initials: LA
authors.lastname: Schneeweis
authors.forename: Ryman
authors.initials: R
authors.lastname: Navoa
authors.forename: Kirby
authors.initials: K
authors.lastname: Steger
authors.forename: Lei
authors.initials: L
authors.lastname: Xie
authors.forename: Christopher
authors.initials: C
authors.lastname: Yonan
authors.forename: Ralph
authors.initials: R
authors.lastname: Abraham
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Grace
authors.forename: Stuart
authors.initials: S
authors.lastname: Lindsay
mesh_terms: D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D058767:Protein Unfolding; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D007074:Immunoglobulin G