Biophysical Studies of the Molecular Motor KinesinKoch Lab, UNM Dept. Physics and Center for High Technology Materials (CHTM)

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

Larry Herskowitz, IGERT FellowPhysics Ph.D. Student

Anthony Salvagno, IGERT FellowPhysics Ph.D. Student

Brigette BlackPhysics Ph.D. Student

Andy Maloney, NSF IGERT FellowPhysics Ph.D. Student

Igor KuznetsovPostdoc

Linh LePhysics B.S. @ UNMNow biophysics grad @ Ohio State

“Kiney”

Brian JoseyPhysics B.S. Student

This talk was presented at the UNM department of chemistry on Sept. 11, 2009. I have tried to give appropriate credit on all images used—Steve Koch

Single-molecule manipulationOptical tweezers; magnetic tweezers; MEMS

Kinesin / mictrotubulesThermostable kinesin; microdevice applications

Protein-DNA interactions; transcription

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

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.

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

Outline for today’s talk

Introduction to kinesin and microtubules

Overview of our DTRA-funded theory / experiment kinesin projectEffects of water on biomolecular interactions

Explanations of assays we use to study kinesin

Early experimental results and modeling!

Kinesin is a eukaryotic molecular motor proteinwith a number of intracellular functions

Mitosis Intracellular transport

Kinesin binds to microtubules and uses ATP hydolysis to walk along tubulin protofilaments

An overview of the two basic components of this system:

Microtubules

Kinesin

Microtubules are a key component of the system:kinesin does not move or catalyze ATP hydrolysisin absence of MTs

Goldstein Lab

25 nm

Microtubules are polymers of tubulin heterodimers

4 nm

8 nm

- + tubulin dimer

Protofilament

25 nm

Microtubules can be reliably polymerized in vitro

In living cells, predominant form of MTs have 13 protofilaments (PFs)

In vitro “reassembly” of microtubules was possible by the early 1970s (Borisy, Brinkley, …)

Typically performed with purified bovine or porcine brain tubulinProduces an assortment of MTs with varying numbers of PFs (usually not 13)Recombinant tubulin is not readily available

MTs are stabilized by taxol … chemical cross-linking is another strategy

Easily visualized by fluorescence microscopy

Kinesin binds to microtubules and uses ATP hydrolysis to walk along tubulin protofilaments

An overview of the two basic components of this system:

Microtubules

Kinesin

Goldstein Lab

Kinesin is a eukaryotic molecular motor proteinwith a number of intracellular functions

MitosisIntracellular transport

Vale, Reese, Sheetz, 1985, Cell 42 39-50. “Identification of a Novel Force-Generating Protein, Kinesin, Involved in Microtubule-Based Motility.”

At least 14 families of kinesin across all eukaryotes

Dimeric “conventional” kinesin-1: vesicle transportKinesin-1, -2, -3, etc…

E.g., Kinesin-5 is tetrameric kinesin: spindle formation

HHMI Winter Bulletin 2005

Kinesin-5 tetramers

Conventional kinesin-1 “walks” along protofiliments in hand-over-hand mechanism

Sablin and Fletterick, 2004 JBC

Processivity

Thorn, Ubersax, Vale JCB 2000

A possible mechanism for kinesin procession

Gray: coiled-coil; blue: catalytic core; white/green: α, β subunits of microtubulin heterodimer; red/orange/yellow: neck linker in successively more tightly-docked states on catalytic core; cargo not shown.

• Step 1: ATP binding to leading head initiates neck-linker docking with catalytic core

• Step 2: Neck-linker docking is completed by leading head, throwing trailing head forward by 16 nm toward next tubulin binding site.

• Step 3: After a random diffusional search, new leading head docks tightly onto the binding site, completing 8 nm motion of attached cargo. Polymer binding accelerates ADP release; trailing head hydrolyzes ATP to ADP-Pi.

• Step 4: ATP binds to leading head following ADP release, and neck-linker (orange) begins to zipper onto catalytic core. The trailing head, which has released its Pi and detached its neck linker (red) from its core, is in the process of being thrown forward.

R. D. Vale and R. A. Milligan, Science 288, 88 (2000).

Truncated, tagged conventional kinesin constructs

Coy, Hancock, Wagenbock, Howard (1999)

Full length conventional kinesin self-inhibits by tail binding to motor domain

Asbury, Fehr, Block (2003)

Recombinant kinesin expressed in E. coli, purified by his-tag methods

Limited commercial availability

Striving for atomistic insights into catalytic mechanism

Sablin and Fletterick, 2004 JBC

Much has been learned about kinesin at the stochastic (mechanical) level

But atomistic understanding of mechanochemistry is lacking

Our goal is to gain atomistic insight via a variety of experiments and simulations

Our DTRA Project: “Coupled Atomistic Modeling and Experimental Studies of Energy Transduction and Catalysis in the Molecular Motor Protein Kinesin”

Susan Atlas and Steve Valone (LANL)“Charge transfer embedded atom model” (CT-EAM)Atomistic modeling of kinesin catalytic core

Kochlab: Biophysical studies of kinesin

An initial connection between theory & experiment:Water!

CT-EAM can correctly model water; can make predictions abouthow osmotic pressure and heavy water will affect kinetics

Kochlab: Can vary water osmotic pressure; heavy / light water

Biophysicists have often ignored water Me too, before I saw work from Parsegian, Rand, Rau

1995

The osmotic stress method relies on changing water activity by adding high concentration of solutes

Parsegian, Rand, Rau, Methods in Enzymology 259 (1995)

“Osmolyte” (sucrose, betaine, PEG, …)Reduces the chemical potential of water

Molecule of interest has a shell of hydrating water molecules

Protein

DNA

Non-specific, Knonsp Specific complex, Ksp

Sidorova and Rau,PNAS 1996

No osmotic stress studies of kinesin untappedUtility proven in protein-DNA studies

Osmotic pressure helpfulFor increasing lifetime too

ln(F

ract

ion

boun

d)

Sidorova and Rau

Osmotic stress dramatically increases lifetime ofbound molecular complexes

Kinesin binding / unbinding

Why is water so important?

Each time the kinesin head binds to tubulin, dozens of “hydrating” water molecules must be excluded.

Each time the kinesin unbinds, water must “rehydrate”

Thus, “water activity” strongly impacts binding kinetics (and whole kinetic cycle)

Okada, Higuchi, Hirokawa

Water excluded

Water hydrating

Osmotic stress increases myosin-actin affinity(only one study I’m aware of)

Highsmith et al. Biophys. J. 1996

No data exist for kinesin-MT

Potentially many high-impact results

So, our first line of experiments will utilize osmotic stress (and heavy water)

Properties of water will provide initial strong ties between theory and experiment

Provide a very interesting line of high-impact experiments

Also provide a connection to technological applications of kinesin / MT systemLong-term stability of kinesin and microtubulesUp-modulation of kinesin processivity? velocity? strength?

We will utilize two independent experimental platforms

“Easy”

Robust

Many experimental “knobs”

Limited readout

More difficult

Many experimental “knobs”

Many readout variables

Gliding motility assay

Andy, Brigette, Linh have gliding assayworking very well in our lab!

Passivated glass surface (casein)

Buffer includes ATP, antifade cocktail

The assay is a bit finicky…light-induced microtubuledisintegration is one problem.

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

Gliding motility assay will be our initial main assay

Operate in the high motor density regime

Main experimental result is transport velocity

Osmotic stress

Light / heavy water

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

Bead motility assay

Adrian Fehr, Chip Asbury Science 2003Steve Block Lab, Stanford

Single-molecule kinesin transportSteve Block Lab, Stanford

Optical Trap“Laser tweezers”

Microsphere

Biomolecular “Tether”

Coverglass

Optical tweezers are formed by shining laser light into a high numerical aperture objective

Kochlab Optical Tweezers

Piezoelectric stage moves coverglass relative to trap center

Infrared laser focused through microscope objective

piezoelectric stage

Quadrant photodiodeto measure force

Optical Trap

Microsphere

Biomolecular “Tether”

Coverglass

Using optical tweezers, we can apply and measure forces on single biomolecules

Newton’s third law

Force on bead = force on lasercollect exit light onto photodiodeto measure force, displacement

Dielectic particles (500 nm polystyrene) attracted to laser focus

Microsphere

Biomolecular “Tether”

Coverglass

Forces from < 1 pN to 100s pN

Length precision ~ 1 nm

Thermal energy (kBT) 4 pN – nm = 1/40 eV

Kinesin 8 nm step, 6 pN stall

RNA Polymerase 0.3 nm step, 25 pN stall

DNA Unzipping 15 pN

Using optical tweezers, we can apply and measure forces on single tethered biomolecules

OT feedback control software is crucial componentWe have a user-friendly LabVIEW application with a variety of feedback modes

Bead motility assay provides wealth of information

High kinesin concentration

Measure velocity of collective molecular motors (similar to gliding assay)

Low kinesin concentration

Single-molecule studies of kinesin: processivityforce-velocitypull-off force

Block et al. (2003) PNAS

Bead motility assay

High kinesin concentration

Measure velocity of collective molecular motors (similar to gliding assay)

Low kinesin concentration

Single-molecule studies of kinesin: processivityforce-velocitypull-off force

Experimental knobs for iterative theory/experiment loop:

Osmotic stress

Light / heavy water

Temperature, metal ions, ATP concentration

Site-directed mutagenesis

We’ve not yet implemented this assay

Modeling: Kinesin kinetic cycle is complicated

Gilbert et al., Nature 1995

R. D. Vale and R. A. Milligan, Science 288, 88 (2000).

We can not measure all of these rate constants directly…We measure: velocity; processivity; stall force; pull-off force

We have written a stochastic simulation for interpreting / predicting results of assays

DT Gillespie, “Exact Stochastic Simulation of Coupled Chemical Reactions” The Journal of Physical Chemistry, V. 8 p. 2340 1977

State machine, written in LabVIEW by Larry

Results of simulation can be viewed with LabVIEW animation – gives insight into kinetic pathway

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

0

50

100

150

200

250

300

350

400

Time (s)

Pos

ition

(nm

)

Simulation of a single kinesin run

0 1000 2000 3000 4000 5000 6000 7000

0

20

40

60

80

100

Run Length (nm)

Num

ber

Repeating many times, we can measure the “processivity” -> average run length

Guydosh @ Block Nature 2009optical tweezers measurements

0 2 4 6 8 10

0

1000

2000

3000

Concentration ATP (mM)

Vel

ocity

(uni

ts?)

Changing ATP concentration in the simulation produces Michaelis-Menten kinetics

200 simulations at each concentration

-1.0 -0.5 0.0 0.5 1.0-100

0

100

200

300

400

500

600

700

Force (units?)

Vel

ocity

(uni

ts?)

Application of opposing and assisting force also reproduces results seen with OT data!

Block et al. (2003) PNAS

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, Brigette Black, Anthony

Salvagno,Linh Le, Brian Josey, Igor Kuznetzov

End

2 3 4 5 6 720

30

40

50

60

1 M Betaine(Sketchy)

0.333 M Betaine

1 M Betaine

Figure 4 Koch SJ and Wang MD

Most pro

bable

un

bin

din

g f

orc

e,

F * (

pN

)

ln (loading rate (pN / s) )

Our preliminary data showed that osmotic stress effects protein-DNA unbinding forces

Specific Non-specific

Diffusing, at sitek-2

k2kdiffkon

k-1 kdiff

X-intercept of these curves reveals off-rateEvans & Ritchie 1997 theory

Protein-DNA interactions probed by DNA unzipping is another Koch Lab project

We anticipate similar effects of osmotic stress on kinesin-MT forced disruption

Kinesin-microtubule unbinding forces

Kawaguchi, Uemura, Ishiwata 2003

Brower-Toland et al., 2002

“Dynamic Strength of Molecular Adhesion Bonds”Evan Evans and Ken Ritchie, 1997 Biophys. J.