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Rotary motors – F 0 F 1 ATPase, helicase Goals – learn about rotary motor basic to biology landmark paper directly observing rotation in F1 ATPase some.

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Presentation on theme: "Rotary motors – F 0 F 1 ATPase, helicase Goals – learn about rotary motor basic to biology landmark paper directly observing rotation in F1 ATPase some."— Presentation transcript:

1 Rotary motors – F 0 F 1 ATPase, helicase Goals – learn about rotary motor basic to biology landmark paper directly observing rotation in F1 ATPase some data from follow-up papers showing more detail over-view of more complex F 0 F 1 “double” rotator coupling proton gradient to ATP synthesis flagellar motors and magnetically-driven  m screws

2 F 1 ATPase Ancient, membrane-associated structure, found in all organisms from bacteria to man, synthesizes or degrades ATP ADP + P Crystal structure shows a central shaft protein (  ) surrounded by a hexamer consisting of 3  and 3   monomers  Many hypotheses that it might act as a rotary motor  coupling rotation to synthesis (degradation) of ATP

3 Crystal structure of mitochondrial F 1 ATPase What is diagram showing? What do you make of the fact that 1 of the  units has ATP, 1 has ADP, and 1 has no nucleotide? (ATP analogue)

4 Experimental set-up – purify component proteins from E Coli How was SA attached to actin and  ? Do either have to be attached at specific sites? How were  units attached to glass? How was actin visualized?

5 What do the pictures show? Is the motion generally unidirectional? Are you sure? What would you expect if the actin were tethered but free to rotate?  t = 33ms

6 Do you expect the slopes to decrease with actin length? How would you expect the slope to vary w/L?  1/L, 1/L 2 ?) Why might slopes differ for filaments of the same length? Why do some traces suddenly flatten out?

7 Brownian equivalents for linear & rotational motion F =  v N =   sphere  = 6  r  sphere = 8  r 3  rod = (  /3)  L 3 /[ln(L/r) – 0.477]  likely underestimate due to  surface proximity and “bumping” on glass  D = k B T/  D r = k B T/   = 6Dt  D r t L = 2000nm, r = 10nm,  = Ns/m 2,  rod  Nms D r  4rad 2 /s, t(1rev=6rad)  1s, t(50rev)  2500s

8 Their conclusions: Rotation in 1 direction when hydrolyzing ATP Torque estimated from velocity and drag coeff (N=  )  30pNnm => more powerful than kinesin, myosin Power (  )   G from ATP hydrolysis with 3 ATP/cycle c/w high efficiency

9 Yasuda et al Nature 410:898 (2001) follow-up paper Why might it be helpful to replace actin with 40nm gold bead? Are you sur- prised bead rotates eccen- trically?  t = 0.5ms

10 Main result: bead pauses (moves) in steps at low [ATP], begin to see 30 0, 90 0 substeps pause before 90 0 step  [ATP] -1 rotations time

11 Goal – relate step-wise motion to binding ATP, hydrolysis of ATP, release of ADP, P i, subunit conformational changes

12 Itoh et al, Nature 427:465 (2004) – magnetically rotating beads in opposite direction -> ATP synthesis Luciferin-luciferase, ADP, P i in buffer; ATP synthesis -> light Compare # photons/5min when rotation clockwise (Syn- thesis), counter-clockwise (Hydrolysis) or no rotation (N)

13 sum Indiv. expts. Main conclusion – Motor is reversible clockwise – synthesize ATP counter-clockwise – hydrolyze ATP Why slt. more light with H than N – a few motors are upside down on top surface of glass! Going cc’wise: think alter subunit affinity for ADP, P i, ATP, etc.

14 Englebrecht Review Nature 459:364 (2009)   Proposed mech. of torque generation compliance coupling stores energy good source of refs to potential presentation papers!

15 But wait, there’s more …. F 1 is part of F 0 F 1 complex F 0 is second rotary motor, embedded in membrane; H + transport thru F 0 drives rotation of  to make ATP

16 Proposed mechanism of ion driven rotation of F 0 - charge aa neut. in membrane

17 Flagellar motor similar to F 0 in that it is membrane- assoc. and driven by ion flow, but many proteins & connected to large, rigid, helical polymer = flagellum Pallen et al, Nature Reviews Microbiology 4:784 (2006) Mechanics of locomotion at low Reynolds # - reciprocal motion doesn’t work, need propellor (Purcell, Am. J. Phys. 45, 3 (1977), PNAS 94:11307 (1997)) Intermittant rotation -> altering flagellar on/off rate allows bacterium to move in direction of food gradient (Berg, Random Walks in Biology, 1993)

18 Man-made version - Magnetically controlled nano-propellors Ghosh et al Nano Letters 9:2243 (2009) SiO 2 propellor w/30nm cobalt evaporated on half of surface random motion without magnet

19 Propell via 3-d orientation-controllable electro magnet generating  50G field at up to 170Hz Propellor advances  210nm/rotation,  40  m/s at 170Hz controlled trajectories; dots = position at different frames

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21 Summary Rotary motors form some of most fundamental bio-nano machines involved in metabolism and locomotion Rotation driven by ion gradients across membranes (F 0 ) Rotation coupled to ATP synthesis from ADP and P (F 1 ) Torques up to 50pNnm Flagellar screw one of few of modes of locomotion at low Reynolds number


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