Conventional kinesin (kinesin-1) transports membrane-bounded cargos such as mitochondria and vesicles along microtubules. In vivo it is likely that several kinesins move a single organelle and it is important that they operate in a coordinated fashion so that they do not interfere with each other. Evidence for coordination comes from in vitro assays, which show that the gliding speed of a microtubule driven by many kinesins is as high as one driven by just a single kinesin molecule. Coordination is thought to be facilitated by flexible domains so that when one motor is bound another can work irrespectively of their orientations. The tail of kinesin-1 is predicted to be composed of a coiled-coil with two main breaks, the “swivel” (380-442 Dm numbering) and the hinge (560-624). The rotational Brownian motion of microtubules attached to a glass surface by single kinesin molecules was analyzed and measured the torsion elasticity constant. The deletion of the hinge and subsequent tail domains increase the stiffness of the motor (8±1 kBT/rad) compared to the full length (0.06±0.01 kBT/rad measured previously), but does not impair motor cooperation (700±16nm/s vs. full length 756±55nm/s - speed in high motor density motility assays). Removal of the swivel domain generates a stiff construct (7±1 kBT/rad), which is fully functional at single molecule (657±63nm/s), but it cannot work in large numbers (151±46nm/s). Due to the similar value of flexibility for both short construct (8±11 kBT/rad vs 7±1 1 kBT/rad) and their different behavior at high density (700±16 nm/s vs. 151±46 nm/s) a new hypothesis is presented, the swivel might have a strain dependent conformation. Using Circular Dichroism and Fluorescence the secondary structure of this tail region was studied. The central part of the swivel is dimeric α-helical and it is surrounded by random coils, thereby named helix-coil (HC) region. Furthermore, an experimental set-up is developed to exert a torque on individual kinesin molecules using hydrodynamic flow. The results obtained suggest for the first time the possibility that a structural element within the kinesin tail (HC region) has a force-dependent conformation and that this allows motor cooperation.

Conventional kinesin (kinesin-1) transports membrane-bounded cargos such as mitochondria and vesicles along microtubules. In vivo it is likely that several kinesins move a single organelle and it is important that they operate in a coordinated fashion so that they do not interfere with each other. Evidence for coordination comes from in vitro assays, which show that the gliding speed of a microtubule driven by many kinesins is as high as one driven by just a single kinesin molecule. Coordination is thought to be facilitated by flexible domains so that when one motor is bound another can work irrespectively of their orientations. The tail of kinesin-1 is predicted to be composed of a coiled-coil with two main breaks, the “swivel” (380-442 Dm numbering) and the hinge (560-624). The rotational Brownian motion of microtubules attached to a glass surface by single kinesin molecules was analyzed and measured the torsion elasticity constant. The deletion of the hinge and subsequent tail domains increase the stiffness of the motor (8±1 kBT/rad) compared to the full length (0.06±0.01 kBT/rad measured previously), but does not impair motor cooperation (700±16nm/s vs. full length 756±55nm/s - speed in high motor density motility assays). Removal of the swivel domain generates a stiff construct (7±1 kBT/rad), which is fully functional at single molecule (657±63nm/s), but it cannot work in large numbers (151±46nm/s). Due to the similar value of flexibility for both short construct (8±11 kBT/rad vs 7±1 1 kBT/rad) and their different behavior at high density (700±16 nm/s vs. 151±46 nm/s) a new hypothesis is presented, the swivel might have a strain dependent conformation. Using Circular Dichroism and Fluorescence the secondary structure of this tail region was studied. The central part of the swivel is dimeric α-helical and it is surrounded by random coils, thereby named helix-coil (HC) region. Furthermore, an experimental set-up is developed to exert a torque on individual kinesin molecules using hydrodynamic flow. The results obtained suggest for the first time the possibility that a structural element within the kinesin tail (HC region) has a force-dependent conformation and that this allows motor cooperation.