Nonlinearly Additive Forces in Multivalent Ligand Binding to a Single Protein Revealed with Force Spectroscopy

Lawrence Livermore National Laboratory, Livermore, California, United States
Langmuir (Impact Factor: 4.46). 03/2006; 22(4):1749-57. DOI: 10.1021/la052087d
Source: PubMed

ABSTRACT 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.

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    • "If a simple linear retraction profile had been assumed such that r = kcv, we would obtain r = 13200 pN s−1, which is a significant deviation from the correct value. The nonlinearity of the extension profile therefore clearly influences the loading rate in our case as reported elsewhere [55,56]. "
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    ABSTRACT: Single molecule force spectroscopy is a technique that can be used to probe the interaction force between individual biomolecular species. We focus our attention on the tip and sample coupling chemistry, which is crucial to these experiments. We utilised a novel approach of mixed self-assembled monolayers of alkanethiols in conjunction with a heterobifunctional crosslinker. The effectiveness of the protocol is demonstrated by probing the biotin-avidin interaction. We measured unbinding forces comparable to previously reported values measured at similar loading rates. Specificity tests also demonstrated a significant decrease in recognition after blocking with free avidin.
    International Journal of Molecular Sciences 12/2012; 13(10):13521-41. DOI:10.3390/ijms131013521 · 2.86 Impact Factor
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    • "Data collection for each force-distance cycle was performed at 2 µm/s, leading to a loading rate of 4 nN/s. For any given experiment, approximately 15,000 force-distance curves were collected, analyzed and fitted to the worm-like-chain model (WLC) [17]. Each experiment was performed at least five times, each time on different blood samples and with different functionalized tips. "
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    PLoS ONE 03/2011; 6(3):e18167. DOI:10.1371/journal.pone.0018167 · 3.23 Impact Factor
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    • "Understanding the contribution of individual bonds to force curves detecting multiple bond ruptures is nontrivial. For parallel bond loading, the unbinding forces of single bonds have been proposed to add both linearly (Florin et al., 1994) or nonlinearly (Ratto et al., 2006). In force spectroscopy studies using purified receptor–ligand pairs, quantized peaks representing multiple single-molecule unbinding events have been observed in the rupture force distribution (Florin et al., 1994). "
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