Motor proteins The definition of motor proteins Miklós Nyitrai, November 30, 2016 Molecular machines key to understand biological processes machines in the micro/nano-world (unidirectional steps): nm, pn use chemical energy (ATP, proton gradient) as fuel to create mechanical work simple biochemical processes can be driven by diffusion and concentration gradients, complex processes more complex machinery regulation of process (directed motion, start-stop, feedback) reliability of process (error correction) The biological motors Biological motors: Work like man-made machines Transport of biological material Copy and translate genetic code Interact with other cells Interact with surroundings Effectuate cell division and motion Collectively enable bacteria to swim Collectively enable muscle to contract Movement in general How do motor proteins fit into larger systems and chategories? Subcellular, cellular levels Requires ATP (energy) Cytoskeleton-mediated Assembly and disassembly of cytoskeletal fibers (microfilaments and microtubules) Motor proteins use cytoskeletal fibers (microfilaments and microtubules) as tracks 1
The kingdom of proteins proteins are the working machines ( DNA is the brain, proteins are the hand ) operate as: enzymes: catalyze chemical reactions ion pump: generate electrical voltage across a membrane and/or create concentration gradients motors: generate mechanical force Cytoskeleton associated proteins A. According to filaments 1. Actin-associated (e.g. myosin) 2. MT- associated (e.g. Tau protein) 3. IF- associated B. According to the binding site 1. End binding proteins ( capping, pl. gelsolin) 2. Side binding proteins (pl. tropomyosin) C. According to function 1. Cross-linkers a. Gel formation (pl. filamin, spectrin) b. Bundling (pl. alpha-aktinin, fimbrin, villin) 2. Polymerization effects a. Induce depolymerization ( severing, pl. gelsolin) b. Stabilizing (pl. profilin, tropomiozin) 3. Motor proteins Common properties of motor proteins 1. They can bind to specific filament types; 2. They can travel along filaments; Why does nature need motors? transport chromosomes positioning during cell division vesicles transport inside the cytoplasm (ingest food, discard waste, deliver proteins) 3. They hydrolyze ATP. endocytosis exocytosis Why does nature need motors? motion (rotation, sliding) rotation or flapping of flagella or cilia in unicellular organisms contraction (muscles) sliding of filaments to produce contraction creation of vesicles (endo/exo-cytosis) cell division more complex machines/processes nucleic acids polymerases (synthesis of RNA, DNA) ribosomes (synthesis of proteins) (ATP production) DNA packaging (compacting DNA inside a virus head) Types of motor proteins A. Linear motors B. Rotary motors 2
Types of motor proteins 1. Actin-based: myosins Conventional (miozin II) and nonconventional myosins Myosin families: myosin I-XVIII 2. Microtubule based motors a. Dynein Flagellar and cytoplasmic dyneins. MW~500kDa They move towards the minus end of MT b. Kinesin Cytoskeletal kinesins Neurons, cargo transport along the axons Kinesin family: conventional kinesins + isoforms. MW~110 kda They move towards the minus end of MT 3. Nucleic acid based DNA and RNA polymerases They move along a DNA and produce force Common properties of motor proteins 1. Structure N-terminal globular head: motor domain, nucleotide binding and hydrolysis specific binding sites for the corresponding filaments C-terminal: structural and functional role (e.g. myosins) 2. Mechanical properties, function In principle: cyclic function and work Motor -> binding to a filament -> force -> dissociation -> relaxation 1 cycle requires 1 ATP hydrolysis They can either move (isotonic conditions) or produce force (isometric conditions). N C The working cycle of motor proteins ATP cycle attached attachment power stroke δ= working distance detachment The duty ratio; processivity Processive motor: r->1 pl. kinesin, DNA-, RNA-polimerase the motor is attached to the track in most of the working cycle detached τ off Duty ratio: back stroke In vitro sliding velocity: v = δ Attached time: = δ v Cycle time: τ total = 1 V Nonprocessive motor: r->0 pl. conventional muscle myosin A motor protein can produce force in the pn range. r = +τ off = τ total δ=working distance (or step size); V=ATPase activity; v=in vitro sliding velocity Linear motors KINESIN STRUCTURE Myosins MYOSIN STRUCTURE 3
The myosin superfamily Myosins move on actin filaments Non-muscle myosins Other linear motor proteins Kinesins and dyneins Move on microtubules 4
head motor domain coupling domain to track (microtubule) dimer ATP binding site tail Kinesin probably the progenitor of all motor proteins cargo binding domain with central linking domain The ATPase cycle of kinesins initial stage ADP bound to both heads one head (leading head) binds weakly (brownian motion) ADP release leads to strong binding; trailing head cannot bind anymore second stage ATP binds to leading head triggers conformational change throwing trailing forward diffusion leads to weak coupling of former trailing head to microtubule (now leading) The ATPase cycle of kinesins third stage ATP hydrolysis by trailing head leading head binds ATP making for a strong binding phosphate release from trailing head and unbinding from microtubule to allow for a next step a few numbers one step: max velocity: max force: mechanical work: 80Å 1 mm/sec (~ 125 steps/sec) 5-6pN efficiency: 40% 4 10-20 J/step The model for kinesin motion Considering one ATP molecule hydrolysis per step, i.e. 12kcal/mol or ~10-19 J/molecule. Kinesin scheme A more detailed scheme 5
Cartoon: a walking kinesin How are they regulated? Roles for kinesins: cell division Roles for kinesins cell elongation chromosome migration http://faculty.plattsburgh.edu/donald.slish/motors.html http://faculty.plattsburgh.edu/donald.slish/motors.html Dynein, cillium The power stroke of the motor protein, dynein, attached to one microtubule, and to a neighboring microtubule causes the fibers to slide past each other (axonemal dynein) The flexible linker protein, nexin, converts the sliding motion to a bending motion Rotary motors Note: the full and accurate mechano-chemical processes not worked out yet but general mechanisms are known. 6
Bacterial flagellar motor F 1 F o ATPase The bacteria flagellar motor The structure of the motor The structure of the motor Vibrio alginolyticus spirillum EM image D. Thomas, N. Francis, and D. Derosier Examples of bacterial flagaella arrangment schemes Another rotary motor: F1F0 ATPase enzyme that catalyzes the formation of ATP from ADP via charge transport F0 part: proton channel F1 part: carries ATP production mechanism of ATP formation by Paul Boyer, UCLA (1973) and structure of the enzyme by John Walker, Cambridge (90 s) (Nobel prize in Chemistry 1997, www.nobel.se) direct experimental verification of mechanism: Kinosita, Nature, 1997 7
F0:membrane bound rotor; rotates upon H+ flux F1:stator; ATP synthesis on beta subunits Alberts, MBOC 100 r.p.s stepper motor (in chloroplasts; 4 protons per ATP) torque 40 pn m (Jung 93, Oster) brownian motion can cause spontaneous rotation can operate in reverse direction (produce a proton gradient by consuming ATP) www.nobel.se no ATP, no net rotation Kinosita, Nature, 1997 Bacteriophage DNA packaging motor 120 steps (90 +30 substeps) Kinosita, Nature, 2001 8
Bacteriophage DNA packaging motor Summary www.purdue.edu 9