Biophysik der Moleküle!

Transcription

1 Biophysik der Moleküle!!"#$%&'()*+,-$./0()'$12$34!4! Molecular Motors:! - linear motors" 6. Dec. 2010!

2 Muscle Motors and Cargo Transporting Motors! There are striking structural similarities but functional differences! Thin Filament! Myosin II! Thick Filament! " Muscle is an organized# structure with many motors! " Can move at high speeds! V=25.000nm/sec! " Cargo transport requires# only one or a few motors! " Moves at lower speeds! V=10nm/sec!

3 A: Linear motors:universelle machinery of locomotion

4 actin filaments! myosin filaments! relaxed! contracted!

5 Muscle Myosin! Taken from Liang et al., JCB 99! N t e r m A!T!P! C t e r m A!T!P! residue #" length (nm)"

6 B: Intracellular Traffic over Long Distances! Axon! 10 µm! See Joe Howard et al. MPI Dresden " Manfred Schliwa et al. LMU!

7 Expolaration of intracellular space by superposition of random walk. Cells test possible use of internalize objects moving them around cytoplasmic spase

8 At what Length Scale is Motility Faster than Diffusion?! Time to swim the body lenth:! s = d/v! Time to diffuse the body lenth:! D = d 2 /D! Einstein: D = k B T/6"#d!! D /! s = (6"#/k B T)vd 2! ecoli" d#10µm" v#100µm/s!! D /! s # 1! Myosin" d#10nm" v#10µm/s!! D /! s << 1! Diffusion " range!

9 Brownian Motion vs. Bacterial Motility!

10 Life at Low Reynolds Numbers:! Swimming in molasses, walking in a hurricane " Dean Astumian! Reynolds number:" R =! d v $% e.g. bacterium! #! #! 10-6 m * 10-5 m/s * 10 3 kg/m 3! 10-3 kg/ms! Thermal noise power:" k P th #! B T! thermal relaxation time! 4*10-21 J! #! s! # 10-8 W! R = 10-5! => No turbulences!" Compare to power of motors:! P mech # W!! See Astumian & Hänggi, Physics Today Nov. 2002, 33-39!

11 Problem! Molecular motors like myosin II work at low Reynolds number,! (i.e. friction forces dominate over inertia forces)! Thermal energy is large compared to the energy of the driving force.! How can a molecular motor work with precision?! 5&()6+(7'$8&9&'*$7')$0':;),$<=$>?@$A=0'&(=*:*$ ATP + H 2 O & ADP + P i 'G= 20kT ATP hydrolysis delivers about 6 * J! A typical motor uses ATP molecule per second.! Typical motor power of order to W" thermal energy : 1kT ~ 4 * J.! typical relaxation time in water ~ s.! i.e. thermal power is of order ~ 10-8 W." its like walking in a hurricane."

12 Myosin-V: A Vesicle Transporter! Cheney et al., Cell, (1993)! " Messenger RNA Transport! " Melanosome Transport! " Transport of Organelles! " Knockout gives dilute! phenotype in mice! " Griscelli$s syndrome!

13 Myosin: a versatile motor Myosin super-family has 19 members common feature: the motor head differences: the anchor domain

14 The Myosin Family! Gaub/Rief/SS 2005! BPM 3.3! 14!

15 the anchor domain determines the function Myosin II-Molecule has long (-helical region and associates into bundles force generation Myosin I has strongly positively charged domains that bind to and stabilize membranes Stabilization of cell membrane Myosin V: processive Motor Transport of chargo

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17 Crossbridges!

18 Myosin-II: The Key Components! Myosin! ATP! ELC! RLC! ATP! 10 nm! ATP! P i! ADP! Aktin! B7+<C.:)DC22$344E$ F@5$G"#3$ "!$

19 The rotating crossbridge model for myosin (Huxley 1957)! 1.) Lymn-Taylor scheme:! Nucleotides regulate attachment and detachment of myosin! 2.) Swinging lever arm hypthesis! 3.) Power stroke model!

20 The Chemo-Mechanical Myosin Cycle! barbed end" pointed end" t s = 2 ms! t c = 30 ms! F@5$G"#3$

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22 Molecular motors are characterized by: t total the cycle time r AV : the duty ratio, i.e. the time on / cycle time the maximal force non-processive processive

23 In Vitro Motility! High Motor Density! Low Motor Density! v=d/t s! v<d/t s! Myosin II is a non processive motor!

24 In vitro motility assay! Kinesin! Myosin!

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26 Kinesin! Myosin!

27 The Current View of Myosin II movement! Vale and Milligan Science (2000)!

28 Crystal Structures Provide a Close Look into the Motor! Vale and Milligan Science (2000)! B7+<C.:)DC22$344E$ F@5$G"#3$ ""$

29 The working stroke is proportional to the lever length!

30 Intensity" Flourescence Energy Transfer (FRET)! Donor" Fluorescence" Acceptor" Fluorescence" Absorbance" Absorbance" Wavelength" How to measure FRET efficiencies:! E = 1! I donor+ acceptor I donoralone Steady state FRET! E = 1! " donor+ acceptor " donor alone Time resolved FRET!

31 Intensity" Flourescence Energy Transfer (FRET)! Donor" Fluorescence" Acceptor" Fluorescence" Absorbance" Absorbance" Wavelength" How to Relate FRET Efficiencies to Distances! E = 1! 1 + # r $ & " r 0 % 6 Efficiency, E! 1.0! 0.8! 0.6! 0.4! 0.2! 0! 20! 40! r 60! 80! 0! distance, r! 100! 120!

32 Conformational Change directly observed! W. Shih et al., Cell (2000)! "H$

33 Time-Resolved FRET Reveals 2 Prestroke States! Prestroke State A! Prestroke State B! Poststroke State B!

34 Optical Tweezers" Laser! Scattered! Ray! F! res! n > n! 0! Trapping for! aperture angles! of > 70 deg.! Diffracted! Ray! 34!

35 Force Feedback! Force! Actin helical # repeat: 36 nm! Distance [nm]! Displacement [nm]! 266! 228! 190! 152! 114! 76! 38! 0! -38! -76! -114! 0.0! 0.2! 0.4! 0.6! Time[s]! 0.8! Digital feedback control!

36 Is myosin a Brownian motor?" step size ranges randomly! from 5.5 und 27.5 nm.! steps are always an integral! number of 5.5 nm.! also backward steps are observed!!

37 @'&I)'J)*$&D$8&()6+(7'$8&9&'*$ " molecular motors diffuse in an energy landscape! " they overcome barriers by thermal excitation! " they can store potential (conformational) energy BUT no kinetic energy.!

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39 Brownian Motors work under " non-equilibrium conditions! A time dependent potential reflects a non-equilibrium situation!

40 Brownian motor under an external force!

41 An artificial thermal ratchet:! Experiment by Libchaber:! Plastic beads-move in a periodically driven laser trap.!

42 Multiple motor proteins are associated with membrane vesicles! Figure 19-26!

43 Kinesin is a (+) end-directed motor!

44 Kinesin!

45 Kinesin Walking S.M. Block, Cell 93, 5 (1998)

46 19.3 Different proteins are transported at different rates along axons! Figure 19-19!

47 19.3 Fast axonal transport occurs along microtubules! Figure 19-20!

48 19.3 Microtubules provide tracks for the movement of pigment granules! Figure 19-21!

49 19.3 Intracellular vesicles and some organelles travel along microtubules! ER! Microtubules! Figure 19-22!

50 19.3 The structure of the kinesin microtubule motor protein! Figure 19-23!

51 Kinesin!

52 19.3 Kinesin is a (+) end-directed motor! Figure 19-24!

53 Kinesin:!

54 Protein-Nanotechnology: " Kinesin motors enable nanoscale transport! H. Hess, G.D. Bachand, V. Vogel! Chemistry, 10 (2004) 2110.!