Negative interference in systems of coupled kinesin: A study of self-assembling complexes with defined structure

Intracellular transport is a crucial process that requires the work of motor proteins to distribute necessary cargos. Many times the motors must move over long distances and against high opposing forces than those generated by single motors. To accomplish this task motors appear to act in teams, as suggested by experiments that show enhanced force production and extended travel lengths. Many motors have been characterized individually, but experiments to study their collective mechanics rely on non-specific groupings where the copy number and geometric arrangement are not explicitly known. In order to resolve the true extent to which each motor contributes enhanced transport properties, a system must be developed that precisely controls the number of motors that are studied. Within this work, a convergent self-assembly approach is presented that allows structurally-defined complexes of kinesin-1 to be created. This approach also provides synthetic control over intermotor spacing and the elasticity of the mechanical motor linkages to rigorously characterize the effects of system structure on the interactions of exactly two motors. This synthetic coupled motor system was then used to examine the extent to which motor grouping enhances the transport properties of cargos. It was determined that the average velocity of coupled kinesin proteins was statistically indistinguishable from that of the single motor, while the average run lengths of the two-motor system were slightly longer (≈ 2X), but less than estimated for a system of non-interacting motors (≈ 4X). This study concludes that, under low loads, intermotor strain in coupled kinesin proteins increases the rate of motor detachment from the microtubule and decreases the rate at which additional motors rebind. The presence of negative interference in these complexes implies that groupings of kinesins preferentially travel in a single motor-attachment state, and that only a subset of cargo-bound motors are used during transport.