Nonlinearly additive forces in multivalent ligand binding to a single protein revealed with force spectroscopy.
We present evidence of multivalent interactions between a single protein molecule and multiple carbohydrates at a pH where the protein can bind four ligands. The evidence is based not only on measurements of the force required to rupture the bonds formed between concanavalin A (ConA) and alpha-D-mannose but also on an analysis of the polymer-extension force curves to infer the polymer architecture that binds the protein to the cantilever and the ligands to the substrate. We find that although the rupture forces for multiple carbohydrate connections to a single protein are larger than the rupture force for a single connection, they do not scale additively with increasing number. Specifically, the most common rupture forces are approximately 46, 68, and 85 pN at a loading rate of 650 +/- 25 pN/s, which we argue corresponds to 1, 2, and 3 ligands being pulled simultaneously from a single protein as corroborated by an analysis of the linkage architecture. As in our previous work polymer tethers allow us to discriminate between specific and nonspecific binding. We analyze the binding configuration (i.e., serial vs parallel connections) through fitting the polymer stretching data with modified wormlike chain (WLC) models that predict how the effective stiffness of the tethers is affected by multiple connections. This analysis establishes that the forces we measure are due to single proteins interacting with multiple ligands, the first force spectroscopy study that establishes single-molecule multivalent binding unambiguously.
Ratto
Timothy V
TV
Chemistry and Materials Science, L-232, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94550, USA. ratto7@llnl.gov
Rudd
Robert E
RE
Langry
Kevin C
KC
Balhorn
Rodney L
RL
McElfresh
Michael W
MW
D003208:Concanavalin A / Q000737:chemistry*; D008024:Ligands; D008358:Mannose / Q000737:chemistry*; D008956:Models, Chemical; D011485:Protein Binding; D013057:Spectrum Analysis
D008024:Ligands; D003208:Concanavalin A; D008358:Mannose