Velocity-dependence of Cargo Loading onto Molecular Shuttles Demonstrates the Glue-like Character of Biotin/Streptavidin

Abstract

Molecular shuttles based on biomolecular motors and their associated filaments are being developed to function as conveyor belts in the molecular factories of the future. An essential design element in these active nanoscale transport systems is cargo loading onto the shuttles. We demonstrate that molecular shuttle velocity has to be optimized to facilitate cargo attachment of nanospheres via biotin-streptavidin linkages. The biotin-streptavidin bond gains its ultimate strength on a timescale of milliseconds due to existence of metastable binding states. As a consequence of the glue-like character of this widely used intermolecular bond, the velocity of molecular shuttles has to be optimized to permit efficient attachment of cargo via biotin-streptavidin linkages.In our experiments, kinesin motor proteins adsorbed to a casein precoated surface were used to propel biotinylated microtubules which were coated with streptavidin at saturating dosages. The microtubule gliding velocity was varied between 50 nm/s and 450 nm/s by changing the kinesin substrate ATP concentration. Finally, biotinylated fluorescein-labeled nanospheres were added in concentrations ranging from 25 pM to 100 pM. Nanospheres attached to the surface and were loaded onto microtubules only as a result of collisions between gliding microtubules and nanospheres. Nanosphere attachment showed an unexpected optimum at an intermediate shuttle velocity.The attachment and detachment processes were modeled by combining rigorous mechanical engineering analysis with detailed physico-chemical models. This contribution will present both, the experimental details of our velocity dependent loading experiments and the theoretical model which explains the optimum on the basis of the complex binding energy landscape of the biotin streptavidin linkages.