Biophysical Studies of the Molecular Motor Kinesin Koch Lab, UNM Dept. Physics and Center for High Technology Materials (CHTM) Steve Koch, DTRA Co-PI, Experimental Lead Asst. Prof. Physics and Astronomy Larry Herskowitz, IGERT Fellow Physics Ph.D. Student Anthony Salvagno, IGERT Fellow Physics Ph.D. Student Brigette Black Physics Ph.D. Student Andy Maloney, NSF IGERT Fellow Physics Ph.D. Student Igor Kuznetsov Postdoc Linh Le Physics B.S. @ UNM Now biophysics grad @ Ohio State “ Kiney” Brian Josey Physics 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 manipulation Optical tweezers; magnetic tweezers; MEMS Kinesin / mictrotubules Thermostable kinesin; microdevice applications Protein-DNA interactions; transcription KochLab Overview / Acknowledgments Susan Atlas —Lead of the DTRA project UNM Physics / Cancer Center / Director of CARC 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. Collaborations Funding DTRA —Basic Science; CHTM —Startup; ACS —Jan Oliver IRG

Single-molecule manipulation

Optical tweezers; magnetic tweezers; MEMS

Kinesin / mictrotubules

Thermostable kinesin; microdevice applications

Protein-DNA interactions; transcription

Outline for today’s talk Introduction to kinesin and microtubules Overview of our DTRA-funded theory / experiment kinesin project Effects of water on biomolecular interactions Explanations of assays we use to study kinesin Early experimental results and modeling!

Introduction to kinesin and microtubules

Overview of our DTRA-funded theory / experiment kinesin project

Effects of water on biomolecular interactions

Explanations of assays we use to study kinesin

Early experimental results and modeling!

Kinesin is a eukaryotic molecular motor protein with 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 hydrolysis in absence of MTs Goldstein Lab

Microtubules are polymers of tubulin heterodimers 25 nm 4 nm 8 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 tubulin Produces 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 - +  tubulin dimer   Protofilament 25 nm

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 protein with a number of intracellular functions Mitosis Intracellular 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 transport Kinesin-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-P i . 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).

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-P i .

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.

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 about how 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

No osmotic stress studies of kinesin  untapped Utility proven in protein-DNA studies Protein DNA Non-specific, K nonsp Specific complex, K sp Sidorova and Rau, PNAS 1996

Osmotic stress dramatically increases lifetime of bound molecular complexes Osmotic pressure helpful For increasing lifetime too ln(Fraction bound) Sidorova and Rau 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 system Long-term stability of kinesin and microtubules Up-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 assay working very well in our lab! Passivated glass surface (casein) Buffer includes ATP, antifade cocktail The assay is a bit finicky…light-induced microtubule disintegration is one problem.

Microtubule velocity in gliding assay is measured via LabVIEW 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 data that 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 2003 Steve Block Lab, Stanford Single-molecule kinesin transport Steve Block Lab, Stanford

Optical tweezers are formed by shining laser light into a high numerical aperture objective Optical Trap “ Laser tweezers” Microsphere Biomolecular “Tether” Coverglass Kochlab Optical Tweezers

Piezoelectric stage moves coverglass relative to trap center Using optical tweezers, we can apply and measure forces on single biomolecules Infrared laser focused through microscope objective piezoelectric stage Quadrant photodiode to measure force Optical Trap Microsphere Biomolecular “Tether” Coverglass Newton’s third law Force on bead = force on laser collect exit light onto photodiode to measure force, displacement Dielectic particles (500 nm polystyrene) attracted to laser focus

Piezoelectric stage moves coverglass relative to trap center

Newton’s third law

Force on bead = force on laser collect exit light onto photodiode to measure force, displacement

Using optical tweezers, we can apply and measure forces on single tethered biomolecules Microsphere Biomolecular “ Tether” Coverglass Forces from < 1 pN to 100s pN Length precision ~ 1 nm Thermal energy (k B T) 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 OT feedback control software is crucial component We have a user-friendly LabVIEW application with a variety of feedback modes

Forces from < 1 pN to 100s pN

Length precision ~ 1 nm

Thermal energy (k B T)

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

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: processivity force-velocity pull-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: processivity force-velocity pull-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

Simulation of a single kinesin run

Repeating many times, we can measure the “processivity” -> average run length Guydosh @ Block Nature 2009 optical tweezers measurements

Changing ATP concentration in the simulation produces Michaelis-Menten kinetics 200 simulations at each concentration

Application of opposing and assisting force also reproduces results seen with OT data! Block et al. (2003) PNAS

Acknowledgments Susan Atlas —Lead of the DTRA project UNM 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. Collaborations Funding DTRA —Basic Science; CHTM —Startup; ACS —Jan Oliver IRG Our Lab —Larry Herskowitz, Andy Maloney, Brigette Black, Anthony Salvagno, Linh Le, Brian Josey, Igor Kuznetzov

End

Our preliminary data showed that osmotic stress effects protein-DNA unbinding forces X-intercept of these curves reveals off-rate Evans & 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 Specific Non-specific

Kinesin-microtubule unbinding forces Kawaguchi, Uemura, Ishiwata 2003 “ Dynamic Strength of Molecular Adhesion Bonds” Evan Evans and Ken Ritchie, 1997 Biophys. J. Brower-Toland et al., 2002