ATP Synthase. Proteins as nanomachines. ATP Synthase. Protein physics, Lecture 11. Create proton gradient across. Thermal motion and small objects

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1 Proteins as nanomachines Protein physics, Lecture 11 ATP Synthase ATP synthase, a molecular motor Thermal motion and small objects Brownian motors and ratchets Actin and Myosin using random motion to go somewhere A molecular motor Very large protein Create proton gradient across cell membrane Used to power cellular processes ATP Synthase Desired chemical reaction Rotation of stalk creates conformation changes of the upper bulb - Initiates chemical reaction Low H+ concentration Outer stalk holds upper bulb stationary High H+ concentration Motion of H+ through pore creates rotation of inner stalk

2 How do we know the thing spins? Too small to see directly (size of protein molecules is smaller than wavelength of visible light) Large fluorescent actin filament added to accentuate t motion Now can be seen optically Kinosita Lab ATP Synthase See quicktime movie Can work in both directions: Proton gradient can spin the stalk in either direction, depending on which side of the membrane has higher concentration Excess ATP can be used to drive proton conduction against gradient F1-ATPase: A Rotary Motor Made of a Single Molecule When ATP was added, the actin filament rotated continuously clockwise (movie). Noji, H. et al., Nature 386, (1997).

3 Stepping Rotation of F1-ATPase at Low ATP Concentrations [ATP] = 20 nm [ATP] = 200 nm At low ATP concentrations, F1 rotates in discrete 120 steps. The stepping rate is proportional to the ATP concentration, indicating that each step is driven by one and only one ATP molecule. In the movie at 20 nm ATP, there is an instant where the F1 motor makes a mistake and steps backward (clockwise). A molecular machine occasionally makes mistakes, and its operation is always stochastic as seen in the figure at left. Because of the stochastic nature, one can never synchronize multiple molecular machines. Making it do what you want Magnetic particle added An external magnetic field can be used to either drive H+ across a cell membrane (eg charging a battery) or to synthesise ATP Thermal motion Large objects are relatively unaffected by thermal motion. Although the atoms may jiggle inside, the object as a whole is unchanged. The picture is very different for nanometer sized objects Here the jiggling can be of the same size as the objects themselves. A small molecule will be continually bumping into its neighbours Brownian motion first observed as the random movement of particles on a fluid (Robert Brown 1827; Jan Ingenhousz 1785) 1889 GL Gouy found that Brownian movement was faster for smaller particles First proper understanding and description came from Einstein 1905

4 Thermal motion A small molecule will be continually bumping into its neighbours This makes specific motion almost impossible its like walking into a hurricane The forces impelling the object in the desired direction are much smaller than the random forces than the random forces exerted by the environment. Order out of chaos the Brownian motor With all this thermal motion taking place you may think it is a wonder that proteins and nanomachines can achieve anything. I th t d ti t h? Is there a way to use random motion to go somewhere? Yes the Brownian motor (Brownian Ratchet) Traditional motor: energy used to cause motion Brownian motor: energy used to cease (or direct) motion Brownian motor: energy used to cease (or direct) motion Thermal motion Brownian motion becomes faster and more important as the size of the particles gets smaller. They have less mass and less inertia and so they are jostled more This difficulty in achieving directed motion is not just applicable to tiny machines, but also to the proteins and small molecules in cells How do proteins achieve anything in their chaotic environments? How do cells survive? Brownian Ratchet Imagine a whole bunch of particles initially confined together When we release them, they will slowly diffuse away in random directions (Pictured in 1D) Now we collect the particles that have moved left and those that have moved right Half have gone left, half have gone right

5 Brownian Ratchet The trick to doing something useful is to find a way to make it more likely for things to go one way than the other. The ratchet is a device designed to do just this. What is the key design element to obtain directed motion? Brownian Ratchet Is this used in protein motion? Eg ATP synthase The reading Making molecules into motors describes the Brownian Ratchet in more detail Brownian Ratchet 1. Start with particles confined by a ratchet potential: one that has a steeper slope on one side than the other 2. Release confinement 3. Turn potential back on 4. Net movement to right Myosin a family of walking motors

6 Myosin a family of walking motors Myosin a family of walking motors Carry a load from one place to another Myosin heads bind to actin filament Load What makes them go forward? Heads Actin filament Myosin walks along Actin Myosin walks along Actin

7 Myosin walks along Actin Motion takes place in two steps Power stroke Random stroke When released swinging freely like when potential is flat When binds is like the potential being turned back on More likely in forward direction than reverse - Still sometimes steps backward Summary Many proteins are nanomachines ATP synthase is a rotary motor Myosin walks hand over hand Are there ways that we can design and build nanomachines for our own purposes? Any nanomachine has to overcome the random thermal motions surrounding it Rather than fighting random motion, nanomachines can make use of it through the principal of the Brownian ratchet Power stroke Random stroke Position of joint created by power stroke provides bias to make forward step more likely l