The document provides an overview of an experimental project studying the motor protein kinesin using various single molecule techniques like optical tweezers and magnetic tweezers. The project is investigating the effects of heavy water (D2O) on kinesin-driven microtubule motility using gliding motility assays. Preliminary results show that microtubules are more stable in D2O, remaining active for over 24 hours compared to regular assays. Velocity measurements of microtubule movement are also being conducted. Future work will explore isotope effects using 18-oxygen water and the effects of osmotic stress on motility. The project involves collaborations between the documenting lab and other groups studying modeling and applications.

1. Experimental overview of DTRA kinesin projectKoch Lab, UNM Dept. Physics and Center for High Technology Materials (CHTM) “Kiney” Steve Koch, DTRA Co-PI, Experimental LeadAsst. Prof. Physics and Astronomy Larry HerskowitzPhysics Ph.D. Student Andy Maloney Physics Ph.D. Student Anthony Salvagno, IGERT FellowPhysics Ph.D. Student Brian Josey Physics B.S. Student SJK Note: For most of the embedded movies, try thispublic directory: http://kochlab.org/files/Movies/2010%20Feb%20DTRA%20Presentation Email: sjkoch@unm.edu Emmalee Jones, (rotating)NSMS Ph.D. Student

2. KochLab Overview / Acknowledgments Single-molecule manipulation Optical tweezers; magnetic tweezers; MEMS Kinesin / mictrotubules Osmotic stress; isotope effects Protein-DNA interactions; transcription Collaborations Susan Atlas—Lead of the DTRA projectUNM Physics / Cancer Center / Director of CARC HaiqingLiu (G. Mantano lab)—Microdeviceapplications of kinesin LANL & Center for Integrated Nanotechnology (CINT) Evan Evans Lab—Single-molecule thermodynamics and kinetics U. New Mexico / U. British Columbia / Boston U. Funding DTRA—Basic Science; CHTM—Startup; ACS—Jan Oliver IRG

3. Microtubules are polymers of tubulin heterodimers Fast polymerizing end Plus end 8 nanometers 25 nanometers Slow polymerizing end Minus end Tubulin can be purified from, for example, cow brains Microtubules can be reliably polymerized in vitro Stabilized with anti-cancer drug Taxol

4. Our goal is to gain atomistic insight through a variety of experiments and simulations…especially focusing on water Susan Atlas (PI) and Steve Valone (LANL) “Charge transfer embedded atom model” (CT-EAM) Atomistic modeling of kinesin catalytic core Molecular dynamics Kochlab: Biophysical experimental studies of kinesin

5. Gliding motility assay Buffer includes ATP, antifade cocktail Andy is currently leading the GMA project 100 microns Passivated glass surface (casein) Parameters we can measure Speedspeed distribution MT morphology (straight; circles; length) Assay longevity (activity; photobleaching) (Show movie externally) “motility in regular water”

6. Gliding motility assay is initially our main assay Buffer includes ATP, antifade cocktail Passivated glass surface (casein) Operate in the high motor density regime Main experimental result is transport velocity Heavy water Osmotic stress Temperature, metal ions, ATP concentration Site-directed mutagenesis Fascinating early results! Experimental “knobs” to obtain datathat can be compared with theory in the iterative loop

7. Heavy water background Naturally abundant 1 / 6600 hydrogen molecules is deuterium 17 mM deuterium in “standard mean ocean water” 11% denser than H2O. Freezes at 3.8C. D-bonds stronger. Toxic to eukaryotes. The toxic effects are similar to chemotherapeutic drugs. D2O has been used to stabilize viral vaccines. D2O stabilizes tubulin and microtubules. D2O stimulates tubulin assembly formation. (Other fascinating factoids…) Effects on kinesin motility has not yet been studied

8. Gliding assays in D2OSquiggly Microtubules; MT-MT interactions (Show movie externally) “Motility in 100% D2O”

9. Gliding assays in D2OSignificantly more stable microtubules (and maybe kinesin) Activity lasts > 24 hours (Show movie externally) (“motility after 1 day in D2O”) Also reduces photobleaching and possibly the “opticution” effects.

10. Microtubule velocity in gliding assay is measured viaLabVIEW image tracking software written by Larry Open source software Preparing publication (Show movie externally)

11. Gliding motility assay—Deuterium Isotope Effects 7. Guydosh, Nicholas R, and Steven M Block. “Direct observation of the binding state of the kinesin head to the microtubule.” Nature 461, no. 7260 (September 3, 2009): 125-128. doi:10.1038/nature08259. Supplemental information. Preparing publication

12. Implications of DTRA research so far Basic Research Gaining insights into fundamental mechanochemistry of kinesin/MTs Properties of water ideal connection with theory group (didn’t show): Have built stochastic simulation to interpret data Applications D2O Results point towards strategies for improving device robustness Fundamental understanding will guide directed engineering of motors Open source software will help community

13. Next steps – Study isotope effect; osmotic stress 18-oxygen water (does not exchange with protein groups; hydrogen bonding same) Osmolytes (e.g. betaine, sucrose) Is this a real effect?

14. Acknowledgments Our Lab—Larry Herskowitz, Andy Maloney, Anthony Salvagno,Brian Josey, Emmalee Jones, Linh Le, Brigette Black, Igor Kuznetsov Collaborations Susan Atlas—Lead of the DTRA projectUNM Physics / Cancer Center / Director of CARC Steve Valone—Co-PI (LANL) Haiqing Liu—Microdevice applications of kinesin LANL & Center for Integrated Nanotechnology (CINT) Evan Evans Lab—Single-molecule thermodynamics and kinetics U. New Mexico / U. British Columbia / Boston U. Funding DTRA—Basic Science; CHTM—Startup; ACS—Jan Oliver IRG

15. End