title: Force spectroscopy of the double-tethered concanavalin-A mannose bond.
abstract: We present the measurement of the force required to rupture a single protein-sugar bond using a methodology that provides selective discrimination between specific and nonspecific binding events and helps verify the presence of a single functional molecule on the atomic force microscopy tip. In particular, the interaction force between a polymer-tethered concanavalin-A protein (ConA) and a similarly tethered mannose carbohydrate was measured as 47 +/- 9 pN at a bond loading rate of approximately 10 nN/s. Computer simulations of the polymer molecular configurations were used to determine the angles that the polymers could sweep out during binding and, in conjunction with mass spectrometry, used to separate the angular effects from the effects due to a distribution of tether lengths. We find that when using commercially available polymer tethers that vary in length from 19 to 29 nm, the angular effects are relatively small and the rupture distributions are dominated by the 10-nm width of the tether length distribution. In all, we show that tethering both a protein and its ligand allows for the determination of the single-molecule bond rupture force with high sensitivity and includes some validation for the presence of a single-tethered functional molecule on the atomic force microscopy tip.
authors.affiliation: Chemistry and Materials Science, Lawrence Livermore National Laboratory, Livermore, California 94550, USA. ratto7@llnl.gov
authors.forename: Timothy V
authors.initials: TV
authors.lastname: Ratto
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Langry
authors.forename: Robert E
authors.initials: RE
authors.lastname: Rudd
authors.forename: Rodney L
authors.initials: RL
authors.lastname: Balhorn
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Allen
authors.forename: Michael W
authors.initials: MW
authors.lastname: McElfresh
mesh_terms: D003198:Computer Simulation; D003208:Concanavalin A / Q000737:chemistry*; D008358:Mannose / Q000737:chemistry*; D013058:Mass Spectrometry; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D036103:Nanotechnology; D011485:Protein Binding
chemical_list: D003208:Concanavalin A; D008358:Mannose