Single molecule mechanical studies of acto-myosin Justin E. Molloy - PowerPoint PPT Presentation

“Single molecule mechanical studies of acto-myosin” Justin E. Molloy Francis Crick Institute LONDON, UK

Why work with individual molecules? • Single molecule experiments can give unequivocal information about how enzymes work and can provide new insights into enzyme mechanism. • Sequential steps that make up biochemical pathways can be observed directly. The chemical trajectory of an individual enzyme can be followed in space and time. • There is no need to synchronise a population in order to study the biochemical kinetics • Single molecule data sets can be treated in a wide variety of ways – e.g. can specifically look for heterogeneity in behaviour (ie strain dependence of rate constants, effects of membrane structure, etc).

Lecture Plan: • What are optical tweezers and how do they work? • Mechanical properties of optical tweezers (picoNewtons and nanometres). • Time-resolution of optical tweezers-based mechanical measurements. • Ultimate sensitivity required to measure mechanical forces produced by individual biological molecular motors (<10k b T). • Single molecule studies of “Motor Proteins” a model system for development of new biophysical methods and especially single molecule approaches. • Allied, laser-based, single molecule methods (TIRF microscopy)

E = mC 2 Momentum, mC = E/C Force = mC/t = P/C (P = optical power) .…calculate the force produced by a 3mW laser pointer….

3-D trap using counter-propagating laser beams Ashkin & Dziedzic, 1971

F scat Single beam “gradient trap” Ashkin et al. 1986 F grad

Laser beam has Gaussian intensity profile. Restoring force is proportional to displacement “Spring-like” F = κ x F x r r = 500 nm, F max = 10 pN Typical: κ = 0.02 pN.nm -1

Dynamic response κ β Stoke’s drag m β = πη 6 r δ δ 2 x x + β + κδ = m x 0 δ δ 2 t t Typical values: κ 1 = m = 5x10 -16 kg f res > 50 kHz π 2 m β = 1x10 -8 N.s.m -1 κ = f < 1 kHz κ ~ 1x10 -5 N.m -1 πβ c 2

1 2 1 1 2 2 Molloy & Padgett (2002) Contemporary Physics 43: 241-258

Move Laser beam very rapidly using Acousto-Optic Device “AOD”

Realistically – things are a bit more complicated!

Thermal motion of an optically trapped particle Thermal noise is ~ 14 nm r.m.s.

Calibrate optical trap stiffness 1) Record thermal noise 2) Apply step displacement

Optical Tweezers

Single molecule experiments: Energy calculations: 1 Photon = 400 pN.nm 1 ATP = 100 pN.nm 1 Ion moving across a membrane = 10 pN.nm Thermal energy (k b T) = 4 pN.nm { 1pN.nm = 1x10 -21 Joules }

SINGLE MOLECULE TECHNOLOGIES: • Some single molecule methods have built-in gain (or signal amplification) – Electrical measurements: – opening of a single ion channel allows thousands of ions to flow across a membrane – this can be measured without greatly affecting the state of the channel – Optical methods: – A single fluorophore can emit millions of photons and output does not (usually) affect the mechanical or chemical properties of the system being studied.

Mechanical Studies no “built-in” gain • Optical Tweezers • Low force regime (e.g. “conformational” changes) • Total spatial control in 3-dimensions • Protein-Protein & Protein-Ligand interactions • MagneticTweezers • Low force regime (only z-axis control) • Ability to apply torque (twist) • DNA topology and DNA-protein interactions • AFM • High force regime (e.g. unfolding) • Imaging (e.g. surface profiling + other methods) • Protein-Protein & Protein-Ligand interactions

SINGLE MOLECULE DATA SETS

k AB A B k BA Transition state theory describes the kinetic E properties of the system A − − e A e e A B ∝ ∝ k T k T k e b k e b AB BA e B ∆ E − − − ∆ B ( e e ) E B A k = = = k T k T AB K e e b b k BA Reaction coordinate

1000 molecules k AB A B k BA Monte Carlo simulation K eq = k AB /k BA k obs =k AB + k BA t (ms)

10 molecules

1 molecule t 1 k BA = 1/t 1 t 2 k AB = 1/t 2

How can we use optical tweezers to understand how molecular motors produce force and movement from ATP?

Filament sliding causes muscle to shorten: Light micrograph myofibril Electron micrograph sarcomere

Acto-myosin ATPase pathway Strong binding states Power-Stroke or Ratchet ? AM AM.ATP AM.ADP.PI AM.ADP AM SLOW M.ADP M M.ATP M.ADP.Pi M Weak binding states RECOVERY STROKE

How do myosin motors actually produce force and movement? Thermal Ratchet or Powerstroke conformational change

Acto-myosin in vitro motility assay : myosin F-actin (S1) ATP ADP+Pi

10 µ m

1 μ m Position Time

Optical trapping of acto-myosin

At HIGH myosin surface density many molecules work together to produce sliding. 300 Displacement (nm) 200 100 0 0 0.5 1 1.5 Time (s)

At LOW myosin surface density single binding interactions become visible. Note: The individual events are “mixed up” with the Brownian noise. But, when myosin binds the VARIANCE falls, this helps identify events.

Basic Analysis (I) t on t off N obs 1/k cat Lifetime distribution gives rate constants time 1/t off 1/t on k cat

Basic Analysis (II) d uni N obs Amplitude Start point is distribution uncertain gives d uni d uni amplitude

Some key findings:

Size of the power-stroke Acto-myosin events scored whenever the data showed a deflection away from the mean. Working stroke is variable and +/- 10nm Events scored each time the variance of the data changed. Working stroke is ~5nm Molloy et al 1995 Nature 378: 209-212

Light chain binding domain (lever arm) determines size of the working stroke Working Stroke (nm) Ruff et al 2001. Nat Struct Biol 8 :226-229 Lever arm length (nm)

Mapping mechanics onto the Acto-myosin ATPase fast slow AM.ADP.PI AM.ADP AM.ADP AM AM.ATP ADP M.ADP.Pi M.ATP 50 nm 0.5 sec

Ensemble Averaging 1) Identify start and end of each event 2) synchronise events dx 2 dx total dx 1 3) Average the event data Phase 1 Phase 2 ADP release ? ATP binding Veigel et al. (1999) Nature 398 :530-533

Ensemble Averaging Members of the myosin I family produce movement in two discrete phases Veigel et al. 1999 Nature 398 :530-533

Both Fast and Slow skeletal muscle myosin also generate movement in two phases Capitanio et al. 2006 PNAS 103 :87-92

Lifetime of the working stroke is load dependent K 1 K 2 push 1.6pN = 55s -1 14s -1 12s -1 10 s -1 pull 1.6pN = Veigel et al. 2003 Nature Cell Biol . 5: 980-986

The myosin family : II VIII XI XII VI VII V III X IX IV I (Tony Hodge, LMB Cambridge)

“ Processive” and “Intermittent” motors • Most myosins and many kinesins interact in an “Intermittent” manner with their track. They must work in teams to produce large movements and forces. • kinesin 1, myosin 5, and most DNA processing enzymes are “Processive” motors and take many steps before detaching from their track. They work as single molecules.

Myosin V Conventional kinesin 36nm 36nm 8nm 0.5 sec Veigel & Molloy Carter & Cross

Myosin 5 walks along actin - 100 taking 36nm steps nm 1 second 36 nm Veigel et al. (2002) Nat. Cell Biol. 4 :59-65.

40 nm 0.5 sec 36 nm per div. 200 ms per div. Veigel et al. (2002) Nat. Cell Biol. 4 :59-65.

How does myosin V walk??…….

Lecture Overview: • Optical Tweezers are relatively simple to build and are compatible with standard laboratory microscopes • They have a sensitivity and time-resolution suitable for studying biological macromolecules and cells • They have contributed to our understanding of the mechanism and function of molecular motors (like kinesin, dynein and myosin) and also of DNA processing enzymes. THE FUTURE……… • The advent of fast cameras, fast parallel processing, and more powerful lasers mean that time-resolution is now in the microsecond regime; and forces of ~100pN are possible opening the possibility to study molecular dynamics and cellular mechanics.