Experimental overview of DTRA kinesin projectKoch Lab, UNM Dept. Physics and Center for High Technology Materials (CHTM)

Steve Koch, DTRA Co-PI, Experimental LeadAsst. Prof. Physics and Astronomy

Larry HerskowitzPhysics Ph.D. Student

Anthony Salvagno, IGERT FellowPhysics Ph.D. Student

Andy MaloneyPhysics Ph.D. Student

“Kiney”

Brian JoseyPhysics B.S. Student

Emmalee Jones, (rotating)NSMS Ph.D. 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

Single-molecule manipulationOptical tweezers; magnetic tweezers; MEMS

Kinesin / mictrotubulesOsmotic stress; isotope effects

Protein-DNA interactions; transcription

Susan Atlas—Lead of the DTRA projectUNM Physics / Cancer Center / Director of CARC

Haiqing Liu (G. Mantano lab)—Microdevice applications of kinesinLANL & Center for Integrated Nanotechnology (CINT)

Evan Evans Lab—Single-molecule thermodynamics and kineticsU. New Mexico / U. British Columbia / Boston U.

TIR Illumination

Magnetic Beads

Computer ControlledElectromagnet

Magnetic FieldGradient ForceF

Single moleculetether (e.g. DNA)

CCD CameraNon-magnetic Aspheric

ScatteredEvanescent Light

TIR Illumination

Magnetic Beads

Computer ControlledElectromagnet

Magnetic FieldGradient ForceF

Single moleculetether (e.g. DNA)

CCD CameraNon-magnetic Aspheric

ScatteredEvanescent Light

Collaborations

Funding DTRA—Basic Science; CHTM—Startup; ACS—Jan Oliver IRG

KochLab Overview / Acknowledgments

Microtubules are polymers of tubulin heterodimers

Tubulin can be purified from, for example, cow brains

Microtubules can be reliably polymerized in vitro

Stabilized with anti-cancer drug Taxol

8 nanometers

25 nanometers

Fast polymerizing endPlus end

Slow polymerizing endMinus end

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

Kochlab: Biophysical experimental studies of kinesin

Molecular dynamics

Gliding motility assay

Passivated glass surface (casein)

Buffer includes ATP, antifade cocktail

Andy is currently leading the GMA project

100 microns

Parameters we can measure

Speed speed distribution

MT morphology (straight; circles; length)

Assay longevity (activity; photobleaching)

(Show movie externally)“motility in regular water”

Gliding motility assay is initially our main assay

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

Experimental “knobs” to obtain datathat can be compared with theory in the iterative loop

Passivated glass surface (casein)

Buffer includes ATP, antifade cocktail

Fascinating early results!

Heavy water backgroundNaturally 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

Gliding assays in D2OSquiggly Microtubules; MT-MT interactions

(Show movie externally)“Motility in 100% D2O”

Gliding assays in D2OSignificantly more stable microtubules (and maybe kinesin) Activity lasts > 24 hours

Also reduces photobleaching and possibly the “opticution” effects.

(Show movie externally) (“motility after 1 day in D2O”)

Microtubule velocity in gliding assay is measured viaLabVIEW image tracking software written by Larry

(Show movie externally)Open source softwarePreparing publication

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

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

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?

Susan Atlas—Lead of the DTRA projectUNM Physics / Cancer Center / Director of CARCSteve Valone—Co-PI (LANL)

Haiqing Liu—Microdevice applications of kinesinLANL & Center for Integrated Nanotechnology (CINT)

Evan Evans Lab—Single-molecule thermodynamics and kineticsU. New Mexico / U. British Columbia / Boston U.

Collaborations

Funding DTRA—Basic Science; CHTM—Startup; ACS—Jan Oliver IRG

AcknowledgmentsOur Lab—Larry Herskowitz, Andy Maloney, Anthony Salvagno,

Brian Josey, Emmalee Jones, Linh Le, Brigette Black, Igor Kuznetsov

End