http://www.google.com/images?rlz=1T4GGLF_enUS278US278&q=von+willebrands+factor+clotting&um=1&ie=UTF-8&source=og&sa=N&hl=en&tab=wi&biw=987&bih=503

Von Willebrands Factor = protein involved in blood clotting, forms large multimers, binds platelets and vessel wall, subject to large shear forces.

How does force affect its interactions?

Novel experimental set-up to study effect of force on interaction between A1 and GPIba

Experimental readout of force at which domains separate

Note they are watching individual moleculesthey can repeatedly test the same molecule

unbinding and rebinding forces are stochastic

Some technical details

How long is linker? 43 amino acids, ~16nm fully extendedHas to be long enough to see rupture

Why use DNA “handles”? To keep proteins off surfaces,separate laser trapped bead from rest of assembly,apply force at single point

How to attach proteins to DNA? via S-S bonds with extraunique cys in protein and SH in synthetic DNA

How does the laser trap work?

Idea: refraction changes direction (momentum) of light

Since momentum is conserved, bead’s momentum changes by – Dp F = -dDp/dt, mainly directed toward

most intense part of beam

Dp

http://www.youtube.com/watch?v=BL9gmMzpRr4&feature=PlayList&p=DB423384C9BB437D&index=1

for more detailed explanation of trap set-up used here

Watch videos at http://tweezerslab.unipr.it or

Optics – counterpropagating laser traps

beamposition

force

Infer bead position from force, knowing stiffness, x=F/k

Relation between force and Dextension Dx = length of “open” A1-linker-GPIba + DNA

- length of A1-GPIba complex + DNA DNA length should be constant at given F If A1 and GPIba domains don’t stretch, Dx due to

extension of flexible aa linker and rot. of A1, GPIba

Bin data by force,show mean andvariance of Dxat rupture or rebinding

Results c/w expected stretch in linker w/force,hence, c/w single tether

How does loading rate, F(t), affect dn of unbinding forces?

E

pulling direction x

DE

Rate of escape at F=0 ~e-DE/kT

For constant pulling force F, energy barrier decreases by FDx, so rate ~e-(DE-FDx)/kT => av. lifetime <t(F)> = <t(0)> e-F(Dx/kT) = e-F /s kT

Dx

analysis to determine relationship between probability of unbinding at force f for a given F(t) and the average lifetime at constant force, t(F), which should depend exp’lly on F

If F changes with time, F(t), dn of unbinding forces changes, and need careful

Dudko-Hummer-Szabo provide this analysis (PNAS 105:15755 (2008))

Relation between p(f) given F(t) and t(F) is:

If data grouped intoforce bins of size DF; hi = p(rupture in ith force bin); in kth bin,F = F0+(k-1/2)DF

evaluated at F=F0+(k-1/2)DF=

calculated <t(F)>

Obs.p(f)

Kim et al observe p(f) at different loading rates, find bimodaldistributions atintermediateloading rates, then calculate<t(F)> for each peakaccording to D-H-S

k1(or2),off = t-1 extrapolated to F=0

2 Note ln t(F) ~ F for eachpeak as in simple theory

Their interpretation:2 different bound states,each with its own exp’ldependence on F (slope s/kT)and predicted zero forceunbinding rate k1,off , k2,off

1/k2,off

1/k1,off

Repeat expts at constant pulling F close to valueat which bound/unbound states are equally likely,watch transitions back and forth B<->UB

From these data calculate prob. of staying in bound state for time t

Two exponentials at intermediate forces, c/w 2 bound states with different unbinding rates (that are fns of F)

straight line => psurvival ~ e-kt ; k = rate of unbindingexp’l survival prob. expected for process with single

random event (like radioactive decay)

Their model: 2 bound states interconvert at (force-dependent) rates k12 and k21 estimated from data

Note both bonds are weakened by force (s>0, “slip” ratherthan “catch”) but conversion to bond with lower koff and smaller s -> tighter binding at high shear (“flex” bond)

Ristocetin (antibiotic) and botrocetin(snake venom toxin) are drugs known to affect vWF

They promoted conversion to state 2 and decreased response to force (s)

Main points

Novel set-up to repeatedly measure interaction between2 protein domains in single protein as function of force

Nice “corroboration” of D-H-S theory that relates unbindingforce distributions at different loading rates to lifetimes at constant force, with complication that…

Their system had bimodal unbinding force distributions atsome loading rates. This suggests protein complex exists in2 different states, each state behaving as expected forsimple model with ln( (t F)) = Fs/kT, but different sensitivities (s’s) for the 2 states

The 2 bound-state model is biologically interesting sinceit leads to a more graded response to force loading foran interaction naturally subject to comparable forces

Will it turn out that many protein interactions have alter-native binding states?

Single-molecule pulling experiments reveal complexities of protein interactions that would be hard to see in bulk assays

Compared to AFM, newer pulling methods allow study inlower force regime (pN vs nN), and away from surfaceswhich can -> artifacts

Relevance to nanotechnology in other areas

methods to detect forces ~0.1-10pN distance changes ~10nm

ways to relate rupture-force distributionsat different pulling rates to lifetimes at constant force

? interest from ME point of view – combining2 “slip” bonds with different force sensitivitiescan mimic “catch” bond (increase in F ->sudden increase in bond strength)