User:Steven J. Koch/Notebook/Kochlab/2009/07/17/DTRA Abstracts/Brigette
Kinesin is a molecular motor that moves within cells. Conventional kinesin (kinesin-1) moves by "walking" along long cylinders of polymerized tubulin molecules, called microtubules. One head of kinesin will bind to a tubulin molecule, release when the other head binds, and then swivel so that it can bind to the next tubulin molecule. This results is one dimensional directed motion along a microtubule, dubbed motility.
In order to move along microtubules, kinesin converts chemical energy into mechanical energy through ATP hydrolysis. With each step, one molecule of ATP is converted to ADP + phosphate. The amount of phosphate produced in ensemble reactions can be measured by adding malachite green, a dye that changes color when it reacts with phosphate, and then measuring absorbance with a spectrophotometer. By measuring the amount of phosphate created in intervals of time, it is possible to deduce kinesin activity, the number of reactions (steps) per second.
The kinetics of kinesin can also be studied with fluoresence microscopy. In what is termed a "gliding motility assay", the long tail of kinesin is bound, leaving the heads untethered. Microtubules are then added, so that the heads of kinesin can bind with the microtubules and push them in directed one dimensional motion. By observing the motion of microtubules under a microscope, the activity and kinetic properties of kinesin can be studied.
If kinesin is to be utilized for commercial and defense purposes, it is of the utmost importance to understand its behavior in a host of environments and reactions to environmental stressors. Currently, the motility and activity of kinesin have been studied mostly in aqueous environments of moderate osmotic pressure. Because each step requires binding and unbinding of extended molecular surfaces, the activity of kinesin should be strongly affected by the water chemical potential and diffusion characteristics. SJK 00:47, 18 July 2009 (EDT)
I am removing the term "environmental stressors" because I'm not sure it has an accepted and clear meaning...please let me know if I'm wrong!
Changing these properties may significantly change the rate, processivity, or stability of kinesin. We are working towards investigating these effects, specifically with deuterium oxide (heavy water) and osmolytes. We will give preliminary results and conclusions regarding the motility and activity of kinesin in these environments by observing microtubules with the ensemble and gliding motility assays.
Steve Koch 00:47, 18 July 2009 (EDT): I think most people would advice against "tipping our hand" at these really cool osmotic pressure and heavy water experiments. However, I think we should stick with our open science goals and I like your abstract. Furthermore, I'm really excited to see how the assays work out!
