Session Topic:
Single Molecule Nano-Biology
Speaker:
Michio Tomishige/The University of Tokyo
Single molecule observations of structural changes in a “walking” motor protein
1. Introduction
Motor proteins are the nanometer-sized molecular machineries that convert chemical energy derived from ATP hydrolysis to mechanical works. Kinesin is a motor protein that moves along a track called microtubule to transport various cargoes inside the cells. Kinesin is a dimeric molecule with two motor domains (called “heads”) that are connected through coiled-coiled stalk. ATP binds to the motor domain and supplies free energy required for motility. The motor domain also has ability to bind to microtubules in an ATP-dependent manner. Its motility mechanism has been the central question in this field and studied extensively since its discovery on 1985.
2. Single molecule techniques
Single molecule technologies have been successfully applied to kinesin research and provided valuable information on its motility. Single kinesin molecules have been directly visualized using total internal reflection fluorescence (TIRF) microscopy, and shown to move processively for more than 1 µm before detaching from the microtubule. Optical trapping bead assay revealed that kinesin moves with regular 8 nm steps and can move against a load of up to 7 pN. These techniques proved to be powerful in characterizing the motility, but still insufficient to uncover the detailed mechanism. Especially, it remained to be answered how kinesin moves and what is the structural basis. To address these issues, another technical advance was necessary that enables to visualize conformational changes at a single molecule level. Here I will introduce our two novel approaches.
3. High-resolution tracking of single moving part
The first question we asked was how kinesin moves. A prevailing model was that kinesin moves in a hand-over-hand manner by exchanging two motor domains (that would cause 16 nm displacement of the rear head). To test this hypothesis, we observed single fluorescent dye attached onto one of the motor heads using FIONA (Fluorescence Imaging One-Nanometer Accuracy) technique that enables to determine centroid position of the dye at the accuracy of ~2 nm [1]. We found that a dye on one of the heads showed stepwise movement of 16 nm, which is twice the step size of kinesin, supporting the hand-over-hand model.
4. Single molecule observation of internal structural changes
Then, what drives the 16 nm displacement of the head? The neck linker, a short stretch that connects motor head with the coiled-coil, has been shown to undergo ATP-dependent conformational changes. This result prompted a model that docking of the neck linker onto the microtubule-bound head moves partner head forward [2]. To test this idea, we observed structural changes of the neck linker using single molecule fluorescence resonance energy transfer (FRET) technique [3]. Donor and acceptor fluorophores are attached before and after the neck linker of one head of a dimer and used to monitor the distance changes between dyes. We found that the neck linker takes two distinct conformations (forward/backward pointing) and that during processive movement neck linker switches between two conformational states every 8 nm steps. These results support the above idea.
Conclusion
We showed that kinesin “walks” by moving two heads alternately, and that the neck linker movement drives/facilitates the displacement of the head. Such a movement should require coordination between two heads, however the mechanism is still unknown. Future investigations using single molecule techniques might help to understand the complete motility mechanism, and also would benefit the studies for regulation mechanism of the molecular motors [4,5].
References
1. A. Yildiz, M. Tomishige, R. D. Vale and P. R. Selvin. Kinesin walks hand-over-hand. Science 303, 676-678 (2004).
2. R. D. Vale and R. A. Milligan. The way things move: looking under the hood of molecular motor proteins. Science 288, 88-95 (2000)
3. M. Tomishige, N. Stuurman and R. D. Vale. Single molecule observations of neck linker conformational changes in the kinesin motor protein. Nat. Struct. Mol. Biol. 13, 887-894 (2006).
4. M. Tomishige, D. R. Klopfenstein and R. D. Vale. Conversion of Unc104/KIF1A kinesin into a processive motor after dimerization. Science 297, 2263-2267 (2002).
5. D. R. Klopfenstein, M. Tomishige, N. Stuurman and R. D. Vale. Role of phosphatidylinositol(4,5) bisphosphate organization in membrane transport by the Unc104 kinesin motor. Cell 109, 347-358 (2002).