User:Brian P. Josey/Notebook/2009/11/09
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Binding of Kinesin Head to Microtubule
I read an interesting article in Nature about the binding state of kinesin to the microtubule. That paper is titled Direct observation of the binding state of the kinesin head to the microtubule by Nicholas Guydosh and Steven Block. It was published on September 3 of this year in Nature, and the article can be accessed from PubMed.
The authors managed to attach a bead to the head of a kinesin, and using an optical trap, they were able to measure directly the movements of the head of the kinesin protein. They did this by attaching the bead to a strand of DNA that was 70 base pairs long, long enough to get out of the way, but short enough to be rigid. The idea was to overcome the limitation of attaching the bead to the head, which gives you only the general location, and the limits of resolution from single-molecule fluorescence techniques.
The observed that when there was an assisting load applied to the kinesin, the steps were broken down into an overshooting motion, ~23 nm forward, and a recovery phase, ~7 nm backward. Together these two phases gave an average stepping distance of 16 nm. Because they could measure the exact time interval of when the kinesin was in these specific states, they could adjust the concentration of ATP to discover how it binds to the heads. Ultimately the confirmed that ATP is bound to the head in both the overshoot and recovery stage of the step.
The order and process that they came up for the stepping is:
They had some concern with the possibility that the load from the optical trap would pull the tethered head up and off of the microtubule. By taking in to the fact that the distance between the bound and transition states is 2 nm, generous, the work was less than 3.4 pN nm. This is lower than the thermal energy of about 4 pN nm. An increase in detachment rate would then be limited by a factor of two to three which is far to small to account for what they were observing.
They found that the unbound head is mobile, where it can be pulled and rotated with the optical trap and the bound kinesin head. They determined that the kinesin will bind both heads to the microtuble until ATP binding in the rear head releases it. They determined that one head will remain behind the other head if the second head is bound to the microtubule. And they propose that both heads will bind to the microtubule only when the neck linker is docked on the rear head and undocked on the front head.