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We have selected eight macromolecular assemblies with obvious moving parts. All of them normally require some energy input: five need ATP, one uses GTP and two are driven by trans-membrane gradients. We want you to describe the various tasks performed by these proteins. Estimate, where possible, how many copies of each protein are found in one the specified cells, how much force they exert on other cellular components, how often they operate and how far they move during each catalytic cycle. We also want you to explain how all these numbers were measured or calculated, and how their energy requirement (where needed) compares with the total ATP turnover in these cells.
Start with text books. For muscle and cytoskeleton see chapter 16 in “Alberts” Molecular Biology of the Cell, or (very much better) chapters 22 and 23 in “Darnell” Molecular Cell Biology (3rd edition). There is a self-assessment test on the cytoskeleton, and some nice pictures at the University
of Texas website. For Bioenergetics see chapter 14 in Alberts and chapter 17 in Darnell. This is a rapidly changing field and it is important to consult the latest editions. You will also find this material useful for the BIOC3390 lectures on bioenergetics and the lectures on muscle and cell motility. Bacterial chemotaxis is covered briefly in Alberts chapter 15 Cell Signalling pages 773 - 778, and in Stryer (1995) Biochemistry 4th edn. chapter 13 Signal Transduction Cascades pages 326 - 332. Apart from the basic mechanism of ribosomal protein synthesis, text books provide less help for the three nucleic acid motors, but there are some excellent reviews which are listed in the general overview below. Several of these are available on line.
This is a team effort, and you should click on the button marked "students" at the bottom of this page find which group you are in. The other buttons lead to webpages devoted to particular motors. Each team should meet well before the tutorial, in order to divide the work between yourselves. Study the editorial by Junge (1999) Proc. Natl. Acad. Sci. USA 96(9), 4735-4737 [available on line in HTML and PDF formats]. Read the general overview which follows before going on to your own specialist section. You must cover several motors if you hope to answer a question on this topic in the final examinations.
X-ray and NMR structures for many of these proteins have been published in the 'Brookhaven' data base. (It moved in 1999.) We have displayed a few in the following pages, but there are plenty more. There is a good search engine for the new site. You could download additional structures, view them with CHIME or RASMOL, and use them for your presentations if you wish.
A surprising amount has been measured on isolated individual protein molecules, but we don’t guarantee that there is a precise answer for the intracellular situation in every case. You may have to make reasonable estimates. It is not necessary to read every paper: we have deliberately given several alternative sources to reduce the pressure on journals, but you might need to go back and locate the original reference quoted in a review. After doing your research, you should meet again to combine your data, and fill any gaps, so that any one of you could present the information on behalf of your group. Prepare some OHP sheets showing the molecular architecture of your motor protein and details of your calculations.
Last year (1998 - 99) several students were surprised to find a question based on this tutorial on their final examination papers. They shouldn't have been surprised: all parts of the course are potentially examinable, and this includes the tutorials. The material was also on two of the lecturers' reading lists. We are not going to tell you the contents of this year's examination paper, which in any case has yet to be approved by your examiners, but there are some lessons to be learned from previous answers:
The question was "Compare and contrast mechanochemical energy transduction in three molecular motors."
The first point is that students had a choice, and could work around some minor gaps in their knowledge, but they needed to know more than just their own presentations. Therefore, listen to your colleagues' presentations in these and other tutorials, and view the other pages of this website.
The second point is that in the final year we expect more than a regurgitation of someone's lecture notes. The key words are "compare and contrast" and we expect you to select relevant material.
We have reproduced below the marking scheme for the previous question. When you are providing factual material in your answers, you should also give some brief indication of the reasons we believe it to be true.
Three motors are required: 25% per molecular motor for relevant factual recall, and 25% for the "compare & contrast" discussion. [The need to subsequently compare and contrast will affect what is relevant.] Experimental evidence may be relevant, but don't labour the point because this isn't a question on laboratory techniques. In some cases the same methods provided mechanistic evidence for more than one motor. By all means mention this, but we would only give credit for a description of "optical tweezers" once.
Kinesin: Linear. Tubulin. Non-reversible. Towards "plus" ends of microtubules. Processive motor. Firm grip - one molecule can move an organelle. Axonal transport. Protein structure: catalytic head groups, load attached to tail. Credit for tubulin structure. Kinesin related proteins with plus-directed and minus-directed variants. Efficiency about 50% i.e. about 40 pN.nm per ATP (say 5pN and 8nm steps, but we don't expect precise recall of step length)
Dynein: Linear. Tubulin. Non-reversible. Towards minus ends. Cilia (structure, doublets and triplets, spokes, sliding and bending) but also cytoplasmic variants. Kinetochores. Two headed / three headed types. Catalytic head groups, load attached to tail. Efficiency about 50%.
Myosin: Linear. Actin. Non-reversible. Towards plus ends. Brief actin attachment in muscle, but what about non-muscle variants? Catalytic cycle: ATP binding detaches head group, then hydrolysis, rebinding to actin, Pi release, power stroke, ADP release. Catalytic head groups, load attached to tail. Control by calcium, but not too much credit for muscle structure since the emphasis is on mechanochemical transduction. Efficiency about 50%, less force but slightly longer steps than kinesin.
ATP synthase: Rotation. Reversible. Large step size. Describe F0 base, stalk and F1 head groups. Binding energy for ATP. Stoichiometry. Energy needed to prise the ATP off the enzyme. Rotating cam shaft. Retaining piece to stop the head group spinning. Visualisation. Efficiency up to 100% under equilibrium conditions.
Flagellar motor: Rotation. Non-reversible, but can change the direction of rotation under physiological control. Small step size. Very high torque. Driven by proton or sodium gradients. Multiple force generating units per motor assembly. Control system with attractants & repellants and methylation, but not too much on this because we asked for mechanochemical transduction.
We would also have accepted GROEL, topoisomerases, DNA gyrase and other motors even though these were not covered in the 1998 - 99 tutorial, but there were no offers from candidates. If you happen to know the facts, and they are relevant to the question - put them in. We will expand the marking scheme to accomodate them.
The Biochemical Society has a new book in the pipeline:
The editors are SJ Higgins & G Banting. You might have heard of them. The full price is £18, but members can buy it for £13:50. There will be copies in the library. It won't be out in time for these tutorials, but it should be out before the exams.
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If you have comments, queries or suggestions, email me at J.A.Illingworth@leeds.ac.uk