User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/05/11/Kinesin and microtubule questions

I have way too many questions about kinesin and microtubules. Thus, I am going to start cataloging them here as a quick reference for myself. Also, once the question is answered, I will post it here.

Questions

• Does kinesin run lengths on microtubules depend on pH? What about if you change the water to heavy water?
• What is the current model for kinesin motility?
  • Hand over hand.
  • Inch worm.
• Could optical tweezers be artificially making kinesin molecules take 8 nm steps?
• Where do kinesin molecules bind on microtubules?
• Does ATP remove a kinesin head from microtubules or, does ATP put a kinesin head on microtubules?
• Will x-rays crosslink microtubules?
• Will storing kinesin in a lot of osmolytes stabilize them?
• Koch scribbles: Model enzyme system for heavy water, osmotic pressure. Glucose oxidase? Catalyase?
• Can we change the speed at which kinesin moves along microtubules by changing the amount of ATP in solution?
• Do kinesin molecules change with age? Thus affecting transport in older cells?
• Will single headed kinesin stay in contact with microtubules because of Van der Wals forces and still "walk" along the µ-tubule?
• What is the ATP turnover rate for kinesin?
• Can kinesin become inactive?
• What are the dimensions of kinesin?
• Why do people use casein to coat coverslips?
  • Answer: Kinesin will denature when it comes in contact with glass. This is why people use casein. Casein is a protein that is amphiphilic and thus will make a bilayer on the glass. Kinesin will then embed itself in the casein substrate if you are doing gliding assays. If you are using bead assays, then the casein is used as a preventative against kinesin coming in contact with glass. Casein is a legacy protein and I'm not sure that people understand why they use it other than it works and that's enough for them.
• Why do we need magnesium in solution for microtubule polymerization?
  • Answer: Check out this for the answers.
• What type of conformation does a 13 protofilament microtubule make?

Things to do

I want to do these simple experiments first to get my hands dirty.

• Make stabilized µ-tubules and write up a procedure etc.
  • Steve Koch 01:11, 17 April 2009 (EDT): Good idea! Let's purchase some tubulin! There are protocols all over the place, of course. Kinesin home page is a good place to start. But I have a bunch of protocols from the Sandia work, which I think is the best place to start. Please be proactive about asking me for protocols. It's been a while (5 years-ish), so I can't easily link to only the "good" ones. But for a start, I started putting some here: \\controller\webpub\files\Protocols\Kinesin, Microtubules , which is available publicly via kochlab.org, e.g. http://kochlab.org/files/Protocols/Kinesin,%20Microtubules
• Determine how to get a 1:1 ratio of kinesin stuck to beads.
• Figure out the Poisson statistics for kinesin attachment and their probability for 2 of them attaching right next to each other on a bead. (Maybe...or maybe I don't care about this one.)