Our knowledge of these proteins is inversely proportional to their molecular weight. In some cases, isolated protein domains have increased our understanding. The smallest motor is kinesin where the mechanics are fairly well understood, followed by the F1ATPase and myosin, and finally dynein, which is huge and something of a mystery. The groups working on kinesin will be able talk about direct measurements with optical tweezers, but the students reporting on dynein must use indirect estimates. Kinesin and cytoplasmic dynein drive the movement of organelles along the tubulin cytoskeleton, but in this tutorial we will mainly study the dynein variant responsible for the whiplash bending movements of sperm tails and cilia.
Kinesin and cytoplasmic dynein are processive motors. There are two head groups, which are thought to function alternately. One head group binds to the tubulin while the other advances to the next binding site. (There are some recent reports of single-headed processive motors, which are difficult to reconcile with this account!) In any event, the motor remains firmly attached to the microtubule for considerable distances, and this enables a single motor to transport large organelles without losing its grip. Myosin in contrast normally binds only briefly to actin during its catalytic cycle, but this brief contact is sufficient because there are about 250 myosin molecules in every thick filament. The structure of the sarcomeres ensures that the actin and myosin do not drift apart.
Try to recall some basic physics: work done = force * distance moved. A force of 1 Newton moving 1 metre requires 1 Joule of energy. So a force of 1 piconewton moving 1 nanometre requires 10E-21 Joules. The free energy of hydrolysis of ATP under typical intracellular conditions is about 50 kJ / mole [6.022 x 10E23 molecules], so each individual molecule of ATP could supply about 8 x 10E-20 Joules. What would be the maximum force * distance for a molecular motor for each ATP split, assuming 100% conversion efficiency from chemical energy into mechanical energy?
Try to put your estimates in context. A typical 70 Kg human being breathes ~10,000 litres of air each day. The exhaled gases contain ~5% CO2 i.e. ~22 moles of carbon in total. If (for example) this energy were derived equally from fats and carbohydrate, we would be looking at a total input of ~11 MJ per day, phosphorylating over 100 moles of ATP (almost 60 Kg!) with an overall conversion efficiency approaching 50%. These are average figures, including tissues which are metabolically inert.
Heart muscle achieves the highest sustained metabolic rate in the human body: these cells contain about 30% mitochondria by volume, and the maximum respiratory rate is 25 micromoles O2/min/g wet muscle. Assume that cardiac muscle, like most tissues, contains ~25% dry matter, most of which is protein. The F1ATPase (ATP synthase) constitutes about 10% of the cardiac mitochondrial protein, but may be only half this figure in mitochondria from other tissues. You may need these figures to put some upper and lower bounds on your estimates, or to check that your answers are reasonable.
The F1ATPase is a rotary system, in contrast to the linear motion achieved by myosin and kinesin. The F0 base piece embedded in the mitochondrial inner membrane is a molecular turbine driven by the trans-membrane proton gradient. Energy is transmitted to the catalytic subunits in the ATP synthase headpiece by a rotating polypeptide camshaft. The rotation of the “cam” distorts the protein subunits, and is used to prise the newly-synthesised ATP off the head piece, to which it is very tightly bound. The energy input is used to drive ATP release, not for bond formation.
It is possible to directly observe the rotation of the F1ATPase using a fluorescence microscope, although considerable ingenuity is required. Noji et al (1997) Nature 386, 299-302 used a genetically modified F1ATPase from a thermophilic bacterium expressed in E. coli. They discarded the F0 basepiece and tethered the F1 motor head groups to a glass plate using polyhistidine tags attached to the N-termini of all three beta subunits. The glass plate had been pre-treated with horseradish peroxidase conjugated with the nickel complex of nitrilotriacetic acid, to which polyhistidine binds with high affinity. [Nitrilotriacetic acid looks like half an EDTA molecule, so it leaves un-coordinated nickel positions available for external ligands.]
The motors were glued down by their large catalytic subunits, leaving the motor shafts exposed, and facing away from the glass. The gamma subunits which form the shaft were modified by site directed mutagenesis to remove the original Cys193 (which is inconveniently far down the shaft) and replace it with serine. These workers also replaced Ser107 in the stalk region with cysteine.
|This single cysteine residue (the only one in the molecule) could then be biotinylated, and linked using streptavidin to fluorescently labelled, biotinylated actin filaments. [Streptavidin has four biotin binding sites.]
The fluorescent actin filaments were many times larger than the tethered motors and could be visualised in a light microscope.
Addition of 2mM ATP caused a small number of the motor shafts marked by the actin filaments to rotate in a counter- clockwise direction. The movie shows the results they obtained.
click the arrow on the
left to start the movie
For rotary motion, the turning force or torque is measured in Newton metres (Nm), and is simply the tangential force turning the shaft (in Newtons) multiplied by the radial distance (in metres) of the force from the axis of rotation. For example, a typical small car engine might achieve 120 Nm at 3000 rpm. To convert this into a power output, remember that 1 Watt is 1 Joule/second, and calculate force * distance moved per second:
Power output = 120 * 2 * 3000/60
= 37.7 kW
The F1ATPase is operating on a somewhat smaller scale! The appropriate torque units are piconewtons * nanometers (pN.nm) and you will need to discover how many copies of the enzyme are present per g of heart, how much oxygen is consumed per minute, and how fast the coupling shafts must spin. How many protons are needed to turn each shaft once? (Remember that there are three catalytic subunits on the headpiece, and about a dozen identical subunits in the stationary part of the basepiece which houses the rotating shaft). How might proton trans-location be coupled to rotary movement?
For dynein in cilia we are looking at a bending movement. All the 9 + 2 microtubules in each axoneme are connected at the basal body, and the dynein pulls one microtubule in the cilium against its neighbour, causing the shaft to bend. The coordinated whiplash movements of the cilia expel the mucus from the lungs. Cilia are very complex structures with over 100 protein components, and are not well understood. Nevertheless you should be able to put some limits on the process. How many cilia per cell? How many mitochondria per cell? How much ATP could each cell manufacture per minute? How many ATP molecules might be consumed per cilium per beat? How many dynein catalytic cycles per beat?
Movement of materials through the cytosol is an important constraint on the performance of living cells. Muscle tissue is adapted to maximise the diffusion of substrates between the mitochondria and the myofibrils, and the groups studying kinesins will encounter the axonal transport of organelles. Motor neurones need to drag many of the major cellular components from their site of synthesis in the cell body near the nucleus down the axon to their point of use at the motor endplate, which in humans may be about 1 metre distant. If you think that is difficult, imagine the task faced by the corresponding cells in a giraffe or a whale. Not only do cells need to export the new material, but they also need to retrieve used or damaged components. Do not forget the need to get the microtubule ‘railway’ itself into position and perform routine maintenance for seventy years when individual items fail.
The problem is obvious in long thin cells like motor neurones, but diffusion is also a constraint in ordinary cuboidal epithelial cells. Each individual cilium has its own internal transport system, because otherwise it would be impossible to get material to the far end fast enough for it to perform its function. There has been sustained selection pressure in this area over the last 2,000,000,000 years: sperm tails are specialised cilia, and are used for a competition where there are no second prizes.
Many common bacteria (including both gram positive and gram negative species) move with the aid of bacterial flagellae. These hollow corkscrew tubes act as simple propellers and are rotated at high speed (up to 100,000 rpm) by powerful molecular motors within the bacterial membrane. In most species the energy to drive these motors comes directly from the transmembrane proton gradient, although some bacteria use a sodium ion gradient instead. Flagellae are rigid structures, but have flexible universal joints (called "hooks") at their base where they insert into the bacterial membrane. Each flagellum contains multiple copies of a single protein called flagellin, but about 50 other genes are necessary for a fully functional motile system.
You are strongly encouraged to read the two reviews on bacterial chemotaxis in the reference list below. In addition, this site has a large section on flagellar motors linked to the "bacteria" button at the bottom of this page, where you will find additional papers on this subject.
The last three motors all involved in nucleic acid metabolism.
Helicase motors are responsible for unwinding double-stranded DNA and RNA during replication, transcription and splicing. Topoisomerases transiently cleave one or both strands of DNA, so that another strand can pass through the break. This is useful for untying knots, and for controlling the degree of supercoiling in the DNA helix. Both topics were reviewed in the 1996 Annual Review of Biochemistry, (see below) and this would be a good place to start. Both of the electronic abstracts have useful links to more recent papers which have cited these reviews, and the full text of both papers can be downloaded in PDF format. Additional papers on both of these topics are available by following the links to the specialised webpages at the foot of this page.
Elongation factor G is a GTP-driven motor that moves the messenger RNA through the ribosome during protein synthesis. Recent work reviewed by Rodnina et al (1999) [see below] suggests that the translocation of the peptidyl-tRNA from the A-site to the P-site is accompanied by large conformational changes in the ribosome. The mechanism has some parallels with that of myosin in that GTP hydrolysis takes place some time before the major re-arrangement takes place. EF-G is a molecular motor that uses the chemical energy from GTP hydrolysis to drive the movement of the mRNA and both tRNAs between their respective ribosomal binding sites.
Many of the following references are available electronically. You can read them and print them out from the computer clusters on the University network. The HTML versions, where available, often contain extra large format diagrams and useful links to related articles, but they will not print as nicely as the PDF files. The PDF versions do not usually have hypertext links, and are intended to look like the original journal articles. There are some oddities to the screen display of PDF files, so the printed PDF version is often better than it looks on screen.
Ashkin, A (1997) Optical trapping and manipulation of neutral particles using lasers Proc. Nat. Acad. Sci. USA 94, 4853-4860. [Click HERE for the HTML version, or HERE for PDF format.]
Amos, LA & Cross, RA (1997) Structure and dynamics of molecular motors Current Opinion in Structural Biology 7(2), 239-46. [No electronic version is available.]
Blair, DF (1995) How bacteria sense and swim Annual Rev. Micro. 49, 489-522.
[Click HERE for the abstract. No electronic full text version is available.]
Brokaw, CJ (1997) Mechanical components of motor enzyme function Biophys. J. 73(2), 938-51.
[Click HERE for the abstract. No electronic full text version is available.]
DeRosier, DJ (1998) The turn of the screw: The bacterial flagellar motor Cell 93, 17 - 20.
[No electronic version is available.]
Endow, SA (1999) Microtubule motors in spindle and chromosome motility. Europ. J. Biochem. 262(1), 12-18. [You will probably need to log in to read this using the usual BIDS password, but an automated system is on the way. Write down the volume and page numbers for future reference. Click HERE for the journal home page, from which you can navigate to the full text PDF version.]
Fisher, ME & Kolomeisky, AB (1999) The force exerted by a molecular motor. Proc. Natl. Acad. Sci. USA 96(12), 6597-6602. [Click HERE for the HTML version, or HERE for PDF format.]
Gittes, F & Schmidt, CF (1998) Signals and noise in micromechanical measurements. Methods In Cell Biology 55, 129-156. [This is a technical account of the experimental techniques. No electronic copies are available.]
Hackney, DD (1996) The kinetic cycles of myosin, kinesin, and dynein Annual Review of Physiology 58, 731-750.
[Click HERE for the abstract. No electronic full text version is available.]
Harada, Y et al (1998) Single molecule imaging and nanomanipulation of biomolecules. Methods In Cell Biology 55, 117-128. [This is a technical account of the experimental techniques. No electronic copies are available.]
Hirokawa, N (1998) Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279(5350), 519-526. [No electronic copies are available.]
Howard J (1997) Molecular motors: structural adaptations to cellular functions Nature 389(6651), 561-7. [Write down the volume and page numbers for future reference. Click HERE for access instructions. Click HERE to gain access via the Leeds University network.]
Hoyt, MA, Hyman, AA & Bahler, M (1997) Motor proteins of the eukaryotic cytoskeleton Proc. Nat. Acad. Sci. USA 94(24), 12747-8. [Click HERE for the HTML version, or HERE for PDF format.]
Huang, JD et al (1999) Direct interaction of microtubule- and actin-based transport motors. Nature 397(6716), 267-270. [Write down the volume and page numbers for future reference. Click HERE for access instructions. Click HERE to gain access via the Leeds University network.]
Khan, S & Sheetz, MP (1997) Force effects on biochemical kinetics Ann. Rev. Biochem. 66, 785 – 804.
[Click HERE for the HTML version, or HERE for PDF format.]
Lohman, TM & Bjornson, KP (1996) Mechanisms of helicase catalysed DNA unwinding Ann. Rev. Biochem. 65, 169 – 214. [Click HERE for the abstract and citations, or HERE for a full-text version in PDF format.]
Lohman, TM et al (1998) Staying on track: Common features of DNA helicases and microtubule motors. Cell 93(1), 9-12. [No electronic copies are available.]
Mehta, AD, Pullen, KA & Spudich, JA (1998) Single molecule biochemistry using optical tweezers FEBS Letters 430, 23–27. [No electronic copies are available.]
Mehta, AD et al (1999) Single-molecule biomechanics with optical methods. Science 283(5408), 1689-1695. [No electronic copies are available.]
Oplatka, A (1998) Are Rotors at the Heart of All Biological Motors? Biochem. Biophys. Res. Commun. 246(2), 301-306. [This is a somewhat idiosyncratic view of molecular motor function. Click HERE for the abstract, or HERE for the full-text PDF version.]
Rodnina MV et al (1999) Dynamics of translation on the ribosome: molecular mechanics of translocation. FEMS Microbiology Reviews 23(3), 317-333. [Click HERE for a full text PDF file.]
Salmon, ED & Tran, P (1998) High-resolution video-enhanced differential interference contrast (VE-DIC) light microscopy. Methods In Cell Biology 56, 153-184. [This is a technical account of the experimental techniques. No electronic copies are available.]
Sheetz, MP (1999) Motor and cargo interactions. Europ. J. Biochem. 262(1), 19-25. [You will probably need to log in to read this using the usual BIDS password, but an automated system is on the way. Write down the volume and page numbers for future reference. Click HERE for the journal home page, from which you can navigate to the full text PDF version.]
Visscher, K & Block, SM (1998) Versatile optical traps with feedback control. Methods In Enzymology 298, 460-489. [This is a technical account of the experimental techniques. No electronic copies are available.]
Wang, JC (1996) DNA Topoisomerases. Ann. Rev. Biochem. 65, 635 – 692. [Click HERE for the abstract and citations, or HERE for a full-text version in PDF format.]
Most electronic journals check your browser IP address and require an institutional subscription to the printed version of the journal. Consequently, many of the hypertext links in this reference list will only work from a university campus computer.
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If you have comments, queries or suggestions, email me at J.A.Illingworth@leeds.ac.uk