User talk:Andy Maloney/Kinesin & Microtubule questions
Magnesium cofactor for GTP?
Steve Koch 16:17, 8 May 2009 (EDT):Can't remember exactly...I actually have a bunch of papers about tubulin catalyzed GTP hydrolysis. Mg is probably required for hydrolysis, but not sure if it's required for GTP binding and polymerization.
Casein
Steve Koch 23:25, 20 April 2009 (EDT): Use of casein in surface tethering assays is part legacy and part magic. I don't know the exact history, but non-fat dried milk (abbreviated NFDM in the literature) has been commonly used in biology for "blocking" surfaces after sticking molecules of interest. For example, in Western blotting, first tranferring the gel, and then "blocking" with NFDM before incubating with the antibody. It's basically a cheap protein. So, maybe that's why it was chosen as a "surface passivation" substance in single-molecule assays. BSA (bovine serum albumin) can also be used. Casein forms micelles in solution, and they are polydisperse. This may help it effectively block surfaces (small micelles fill in gaps between big micelles). In the case of gliding motility assays, it seems to have some "magic" properties in that it keeps the kinesin molecules active, but I don't think anyone knows why. In general, specific preparation of the surfaces would be much better than use of casein. On the other hand, casein works. I have a whole bunch of casein literature somewhere, about the micelles and whatever.
A couple answers
Steve Koch 00:00, 14 April 2009 (EDT):
- Definitely hand-over-hand. (And I now realize more irony that they call the feet heads, and use the hand-over-hand model.)
- How OT are affecting it is a good question--but there are lots of data saying that it's not. E.g. single-molecule tracking without an OT.
- The osmolyte storage idea that arose during our discussion today has huge potential. Do you still have the window open to the paper we saw that made us think of that? (Something about osmotic pressure stabilizing some other proteins.)
- This can't be our priority...but it would be cool to repeat those kinesin aggregation experiments I did (dynamic light scattering) in the presence of huge omsotic pressure.
Steve Koch 03:46, 16 April 2009 (EDT):
- The Koch scribbles question is answered in the more recent Meth Enzym Parsegian review
- Maximum ATP turnover rate (at saturating ATP and microtubule concentration; that is, >> than the Km for those substrates) is known for many kinesins. It can be measured via bulk or ensemble assays that measure ATP turnover. We used the "malachite green" assay (see Susan Gilbert lab papers) at Sandia because we were interested in doing elevated temperature studies. The couple-enzyme assay (see kinesin home page) is much easier.
- I think ionic association is likely more important than vanderwaals for the weak association of kinesin that could account for processivity of single-headed kinesin. I didn't re-read it, but check this paper in my directory: Biophys J 84_01833_Lakamper et al_Single Fungal Kinesin processivity.pdf . Or probably this is better: nature 424_01804_Hirokawa_Processivity KIF1A.pdf
