Characterization and cooperative dynamics of kinesin motor proteins in intracellular transport

Intracellular transport is typically carried out by teams of motor proteins rather than by isolated molecules, yet the rules governing multi-motor coordination remain poorly understood because motor number, spacing, and composition are difficult to control experimentally. In this dissertation, I developed both the molecular engineering and nanostructural framework needed to address this problem. I designed a modular plasmid toolkit for controlled kinesin expression and purification, and I created a novel DNA origami scaffold that allows motors to be positioned with defined stoichiometry and geometry on a rigid cargo-like platform. Together, these advances provided a programmable experimental system for dissecting how kinesin-1 ensembles and mixed kinesin-1/kinesin-14 assemblies behave under well-defined structural conditions.Using this platform, I found that motor valency strongly enhances attachment persistence and transport robustness, but that the magnitude and interpretation of this effect depend on nucleotide state and ensemble architecture. In weak-binding ADP conditions, increasing kinesin-1 copy number strongly stabilized microtubule association, revealing that additional motors provide a disproportionate rescue benefit in vulnerable binding regimes. In ATP, processivity increased with motor number while velocity changed comparatively little, but the motor-number dependence was more geometry- and preprocessing-sensitive, motivating statistical and modeling analysis. Direct mean-field modeling captured the ADP results reasonably well but proved fragile for ATP, whereas a microstate continuous-time Markov chain model with a global attachment scale regularized this instability and supported an ensemble-rescue interpretation. Finally, introducing antagonistic kinesin-14 reduced kinesin-1-driven transport and produced tunable mixed-motor behavior, showing that this engineered platform can reveal how motor copy number, geometry, and opposition collectively regulate cargo transport. These findings have broader significance for understanding transport defects in neurodegenerative and other trafficking-related diseases, where failures in motor coordination and cargo delivery can have major cellular consequences. More broadly, the ability to engineer and quantitatively tune multi-motor assemblies may inform future bioengineering efforts in nanoscale transport design, including programmable cargo handling and motor-based drug delivery systems.