  With their longest dimension typically being less than 100 nm, molecular
motors are significantly below the optical-resolution limit. Despite
substantial advances in fluorescence-based imaging methodologies, labeling with
beads remains critical for optical-trapping-based investigations of molecular
motors. A key experimental challenge in bead-based assays is that the number of
motors on a bead is not well defined. Particularly for single-molecule
investigations, the probability of single versus multiple-motor events has not
been experimentally investigated. Here, we used bead travel distance as an
indicator of multiple-motor transport and determined the lower-bound
probability of bead transport by two or more motors. We limited the ATP
concentration to increase our detection sensitivity for multiple- versus
single-kinesin transport. Surprisingly, for all but the lowest motor number
examined, our measurements exceeded estimations of a previous model by R2-fold.
To bridge this apparent gap between theory and experiment, we derived a
closed-form expression for the probability of bead transport by multiple
motors, and constrained the only free parameter in this model using our
experimental measurements. Our data indicate that kinesin extends to ~57 nm
during bead transport, suggesting that kinesin exploits its conformational
flexibility to interact with microtubules at highly curved interfaces such as
those present for vesicle transport in cells. To our knowledge, our findings
provide the first experimentally constrained guide for estimating the
probability of multiple-motor transport in optical trapping studies. The
experimental approach utilized here (limiting ATP concentration) may be
generally applicable to studies in which molecular motors are labeled with
cargos that are artificial or are purified from cellular extracts.
