Experimental Overview Koch Lab, UNM Dept. Physics and Center for High Technology Materials (CHTM)

Steve Koch, 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. Student

“Kiney”

SJK: This is a talk I gave for group meeting summer 2009…please let me know if images or other need attribution

Experimental group expertise

Single-molecule manipulationOptical tweezers; magnetic tweezers; MEMS

Kinesin / mictrotubulesThermostable kinesin; microdevice applications

Protein-DNA interactions; transcription

Collaborations:

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

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

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

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 hydolysis 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

We will utilize two independent experimental platforms

“Easy”

Robust

Many experimental “knobs”

Limited readout

More difficult

Many experimental “knobs”

Many readout variables

KinesinKinesin

MicrotubuleMicrotubule

GlassGlassSurfaceSurface(passivated(passivatedwith casein)with casein)

+ Buffer, ATP+ Buffer, ATP

Gliding motility assay

Gliding motility assay, Koch @ SandiaThermophilic fungal kinesin(field of view approx 150 microns)Microtubule velocity is measured either

manually or by automatically tracking MT ends

Larry Herskowitz (grad) is currently adapting existing tracking software for this purpose

Image: George Bachand

KinesinKinesin

MicrotubuleMicrotubule

GlassGlassSurfaceSurface(passivated(passivatedwith casein)with casein)

+ Buffer, ATP+ Buffer, ATP

Gliding motility 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

Gliding motility assay, Koch @ SandiaThermophilic fungal kinesin(field of view approx 150 microns)

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

Image: George Bachand

Bead motility assay

Andrian Fehr, 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

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

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

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.

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

Our initial experiments will pursue effects of osmotic stress and light / heavy water in two experimental assays

Water connects our experiment / theory iterative loop

In addition to coupling experiment / theorythese are an exciting, untapped line of experiments

Only a couple papers exist for myosin / none for kinesin

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

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 (higher chemical potential)

Osmotic stress increases myosin-actin affinity

Highsmith et al. Biophys. J. 1996

No data exist for kinesin-MT

Potentially many high-impact results

Protein

DNA

Non-specific, Knonsp Specific complex, Ksp

Sidorova and Rau,PNAS 1996

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

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

Next up, Susan will describe novel theoretical methods and look at the impact of these experimental “knobs”

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?