Kinesin-1 is a molecular motor that transports cellular cargo along microtubules by completing hundreds of consecutive steps of its two head domains. Questions remain over what conformational changes drive this ATP-powered motility, and how actions of the two heads are coordinated. Photonic Force Microscopy allows optical trapping and 3D position detection of particles, including their axial position, which is often ignored in similar experimental set-ups. Using this technique, the energy landscape of a bead, tethered to a microtubule by a single full-length kinesin molecule on the bead surface, was analysed in the presence and absence of nucleotides. The mechanical properties of kinesin molecules were extracted from these data and were found to alter significantly depending on whether no nucleotide or the ATP analogue AMP-PNP was present in the experimental chamber. The effective axial stiffness of the molecule away from the microtubule increased on AMP-PNP binding. A further increase in this stiffness associated with a decrease in the volume accessible to the bead along the kinesin axis was observed and attributed to a change from 1- to 2-headed microtubule binding. Simulations of a model kinesin structure, consisting of rigid rods and flexible hinges, undergoing thermal fluctuations were performed. They suggest a change in angular stiffness and orientation of the neck domain would explain the change in axial stiffness observed. Neck- linker docking on AMP-PNP binding and inter-head strain caused by two-headed microtubule attachment can account for both these structural changes. Taken together, the experimental and simulated results suggest that when the rear kinesin head has its neck linker docked, and both heads are microtubule-bound, the neck and lower half of the kinesin stalk become aligned parallel to the microtubule, reducing the cargo's height above the surface by 20 nm. Further analysis of the processive motion of kinesin in a saturating concentration of ATP, as is present in physiological conditions, together with these static results led to a full description of the motor movement with respect to chemical and conformational states. This description provides a novel explanation of how the molecule might detect useful movements of its cargo, caused by thermal forces, and use these to increase its efficiency. In this way the motor acts like a mechanical feedback loop, amplifying neck linker dynamics. Similarly, positive movements of its cargo caused by the pull of another molecule motor could be used to aid procession. This additionally explains cooperativity between molecular motors. The model developed also incorporates the presence of a state in which a kinesin head is weakly associated to a microtubule by what is thought to be an electrostatic attraction when the head has ADP bound. This state was unambiguously documented experimentally in this work for the first time, and allows a kinesin head to slide along a microtubule protofilament while only weakly feeling the periodic potential of the ab-tubulin subunits. The significance of this weak association is discussed in terms of how it might aid kinesin motility, including the procession of single-headed constructs, and how it may play a role in the regulation of kinesin function in a cell according to chemical stimuli or spatial position.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:551298 |
| Date | January 2011 |
| Creators | Jeppesen, George |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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