Multivalent Protein Binding Kinetics of Variable Lymphocyte Receptors Probed using Dynamic Force Spectroscopy

Abstract

The binding of an antibody to a target antigen underpins an array of biological warfare agent detection schemes and pharmaceutical therapies. However, current antibody-based detection and treatments suffer from multiple drawbacks, including the lack of an antibody that offers a range of affinities for a target antigen. Here, we measure the binding kinetics of a recently discovered class of antibody derived from jawless vertebrates called Variable Lymphocyte Receptors (VLRs), which can achieve IgG-like binding specificity. We first tether low densities of mono-, di-, and pentavalent VLRs to an atomic force microscope cantilever via polyethylene glycol linkers, and then measure the forces required to rupture their bonds with a substrate tethered anthrax antigen. We find that in all three cases the interactions are load-rate dependent - probed far from equilibrium - over the entire range of load rates for which our apparatus is capable. As expected, binding affinity is enhanced in a noncooperative fashion with increasing VLR valency. Furthermore, for the di- and pentavalent molecules, the at-surface dwell time is varied, with longer dwell times increasing the probability of states in which multiple VLR binding domains of a single VLR protein are complexed with surface bound BclA. Thus, through single molecule force spectroscopy, VLRs with greater valency show increased binding stability and lower dissociation rates, and therefore tunable affinity, while the nature of how the binding state probabilities vary with time provides insight into the intermediate binding state kinetics of this system.