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Regulation; Molecular Motors I

Regulation; Molecular Motors I. Andy Howard Introductory Biochemistry 24 November 2014. Regulation and motors. Even though it isn ’ t really an enzyme, hemoglobin can teach us how allostery in enzymes works. After that we ’ l l talk about molecular motors. Molecular Motors Microtubules

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Regulation; Molecular Motors I

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  1. Regulation;Molecular Motors I Andy HowardIntroductory Biochemistry 24 November 2014 Hemoglobin; Molecular Motors I

  2. Regulation and motors • Even though it isn’t really an enzyme, hemoglobin can teach us how allostery in enzymes works. • After that we’ll talk about molecular motors. Hemoglobin; Molecular Motors I

  3. Molecular Motors Microtubules Tubulin Structure Cilia & flagella Axonemes Dynein, Kinesin Globins Tertiary structure Quarternary structure R and T states Allostery Bohr effect BPG as an effector Sickle-cell anemia Globins & Motor Topics Hemoglobin; Molecular Motors I

  4. Myoglobin structure Sperm whale myoglobin; 1.4 Å18 kDa monomerPDB 2JHO • Almost entirely -helical • 8 helices, 7-26 residues each • Bends between helices generally short • Heme (ferroprotoporphyrin IX) tightly but noncovalently bound in cleft between helices E&F • Hexacoordinate iron is coordinated by 4 N atoms in protoporphyrin system and by a histidine side-chain N (his F8): fig.15.25 • Sixth coordination site is occupied by O2, H2O, CO, or whatever else fits into the ligand site Hemoglobin; Molecular Motors I

  5. O2 binding alters myoglobin structure a little • Deoxymyoglobin: Fe2+ is 0.55Å out of the heme plane, toward his F8, away from O2 binding site • Oxymyoglobin: moves toward heme plane—now only 0.26Å away (fig.15.26) • This difference doesn’t matter much here, but it’ll matter a lot more in hemoglobin! Hemoglobin; Molecular Motors I

  6. Hemoglobin structure • Four subunits, each closely resembling myoglobin in structure (less closely in sequence);H helix is shorter than in Mb • 2 alpha chains,2 beta chains Human deoxyHb65kDa heterotetramerPDB 2HHB, 1.74Å Hemoglobin; Molecular Motors I

  7. Subunit interfaces in Hb • Subunit interfaces are where many of the allosteric interactions occur • Strong interactions:1 with 1 and 2,1 with 1 and 2 • Weaker interactions:1 with 2, 1 with 2 Image courtesy PittsburghSupercomputingCenter Hemoglobin; Molecular Motors I

  8. Subunit dynamics • 1-1 and 2-2 interfaces are solid and don’t change much upon O2 binding • 1-2 and 2-1 change much more:the subunits slide past one another by 15º • Max movement of any one atom ~ 6Å • Residues involved in sliding contacts are in helices C, G, H, and the G-H corner • This can be connected to oxygen binding and movement of iron atom toward the heme plane Hemoglobin; Molecular Motors I

  9. Conformational states • We can describe this shift as a transition from one conformational state to another • The stablest form for deoxyHb is described as a “tense” or T state • Heme environment of beta chains is almost inaccessible because of steric hindrance • That makes O2 binding difficult to achieve • The stablest form for oxyHB is described as a “relaxed” or R state • Accessibility of beta chains substantially enhanced Hemoglobin; Molecular Motors I

  10. Hemoglobin allostery • Known since early1900’s thathemoglobin displayedsigmoidal oxygen-binding kinetics • Understood now to be a function of higher affinity in 2nd, 3rd, 4th chains for oxygen than was found in first chain • This is classic homotropic allostery even though this isn’t really an enzyme Hemoglobin; Molecular Motors I

  11. R  T states and hemoglobin • We visualize each Hb monomer as existing in either T (tight) or R (relaxed) states; T binds oxygen reluctantly, R binds it enthusiastically • DeoxyHb is stablest in T state • Binding of first Hb stabilizes R state in the other subunits, so their affinity is higher Hemoglobin; Molecular Motors I

  12. Binding and pO2 • Hill found that that binding could be modeled by a polynomial fit to pO2 • Kinetics worked out in 1910’s: didn’t require protein purification, just careful in vitro measurements of blood extracts Sir Archibald V. Hill photo courtesy nobelprize.org Hemoglobin; Molecular Motors I

  13. Hill coefficients • Actual equation is on next page • Relevant parameters to determine are P50, the oxygen partial pressure at which half the O2-binding sites are filled, and n, a unitless value characterizing the cooperativity • n is called the Hill coefficient. Hemoglobin; Molecular Motors I

  14. pO2 and fraction oxygenated • If Y is fraction of globin that is oxygenated and pO2 is the partial pressure of oxygen,then Y/(1-Y) = (pO2 /P50)n • 4th-edition formulation: P50nK soY/(1-Y) = pO2n / K • P50 is a parameter corresponding to half-occupied hemoglobin (Y=1/2) • work out the algebra: • When pO2 = P50, Y/(1-Y) = 1n=1 so Y = 1/2. • Note that the equation on p.496 of the enhanced 3rd edition is wrong! Hemoglobin; Molecular Motors I

  15. Real Hill parameters (p.503) • Human hemoglobin has n ~ 2.8, P50 ~ 26 Torr • Perfect cooperativity, tetrameric protein: n =4 • No cooperativity at all would be n = 1. • Lung pO2 ~ 100 Torr;peripheral tissue 10-40 Torr • So lung has Y~0.98, periphery has Y~0.06! • That’s a big enough difference to be functional • If n=1, Ylung=0.79, Ytissue=0.28; not nearly as good a delivery vehicle! Hemoglobin; Molecular Motors I

  16. MWC theory • Monod, Wyman, Changeux developed mathematical model describing TR transitions and applied it to Hb • Accounts reasonably well for sigmoidal kinetics and Hill coefficient values • Key assumption:ligand binds only to R state,so when it binds, it depletes R in the TR equilibrium,so that tends to make more R Jacques MonodPhoto Courtesy Nobelprize.org Hemoglobin; Molecular Motors I

  17. Koshland’s contribution • Conformational changes between the two states are also clearly relevant to the discussion • His papers from the 1970’s articulating the algebra of hemoglobin-binding kinetics are amazingly intricate Dan KoshlandPhoto Courtesy U. of California Hemoglobin; Molecular Motors I

  18. Added complication I: pH • Oxygen affinity is pH dependent • That’s typical of proteins, especially those in which histidine is involved in the activity (remember it readily undergoes protonation and deprotonation near neutral pH) • Bohr effect (also discovered in early 1900’s): lower affinity at low pH (fig. 15.33) Christian Bohrphoto courtesyWikipedia Hemoglobin; Molecular Motors I

  19. How the Bohr effect happens • R form has an effective pKa that is lower than T • One reason: • In the T state, his146 is close to asp 94. That allows the histidine pKa to be higher • In R state, his146 is farther from asp 94 so its pKa is lower. Cartoon courtesy Jon Robertus, UT Austin Hemoglobin; Molecular Motors I

  20. Physiological result of Bohr effect • Actively metabolizing tissues tend to produce lower pH • That promotes O2 release where it’s needed Hemoglobin; Molecular Motors I

  21. CO2 also promotes dissociation • High [CO2] lowers pH because it dissolves with the help of the enzyme carbonic anhydrase and dissociates:H2O + CO2 H2CO3 H+ + HCO3- • Bicarbonate transported back to lungs • When Hb gets re-oxygenated, bicarbonate gets converted back to gaseous CO2 and exhaled Hemoglobin; Molecular Motors I

  22. Role of carbamate • Free amine groups in Hb react reversibly with CO2 to form R—NH—COO- + H+ • The negative charge on the amino terminus allows it to salt-bridge to Arg 141 • This stabilizes the T (deoxy) state Hemoglobin; Molecular Motors I

  23. Another allosteric effector • 2,3-bisphosphoglycerate is a heterotropic allosteric effector of oxygen binding • Fairly prevalent in erythrocytes (4.5 mM); roughly equal to [Hb] (~2.2mM) • Hb tetramer has one BPG binding site • BPG effectively crosslinks the 2  chains • It only fits in T (deoxy) form! Hemoglobin; Molecular Motors I

  24. BPG and physiology • pO2 is too high (40 Torr) for efficient release of O2 in many cells in absence of BPG • With BPG around, T-state is stabilized enough to facilitate O2 release • Big animals (e.g. sheep) have lower O2 affinity but their Hb is less influenced by BPG Hemoglobin; Molecular Motors I

  25. Fetal hemoglobin • Higher oxygen affinity because the type of hemoglobin found there has a lower affinity for BPG • Fetal Hb is 22;  doesn’t bind BPG as much as . • Fetal Hb has more pronounced Bohr effect • That helps ensure that plenty of O2 gets from mother to fetus across the placenta Human fetal hemoglobin65kDa heterotetramer2.5Å Hemoglobin; Molecular Motors I

  26. Sickle-cell anemia • Genetic disorder: Hb residue 6 mutated from glu to val. This variant is called HbS. • Results in intermolecular interaction between neighboring Hb tetramers that can cause chainlike polymerization • Polymerized hemoglobin will partially fall out of solution and tug on the erythrocyte structure, resulting in misshapen (sickle-shaped) cells • Oxygen affinity is lower because of insolubility Hemoglobin; Molecular Motors I

  27. Sickling and polymerization Hemoglobin; Molecular Motors I

  28. Why has this mutation survived? • Homozygotes don’t generallysurvive to produce progeny;but heterozygotes do • Heterozygotes do have modestly reduced oxygen-carrying capacity in their blood because some erythrocytes are sickled • BUT heterozygotes are somewhat resistant to malaria, so the gene survives in tropical places where malaria is a severe problem Deoxy HbS2.05 ÅPDB 2HBS Hemoglobin; Molecular Motors I

  29. How is sickling related to malaria? • Malaria parasite (Plasmodium spp.) infects erythrocytes • They’re unable to infect sickled cells • So a partially affected cell might survive the infection better than a non-sickled cell • Still some argument about all of this • Note that most tropical environments have plenty of oxygen around (not a lot of malaria at 2000 meters elevation) Plasmodium falciparumfrom A.Dove (2001) Nature Medicine 7:389 Hemoglobin; Molecular Motors I

  30. Other hemoglobin mutants • Because it’s easy to get human blood, dozens of hemoglobin mutants have been characterized • Many are asymptomatic • Some have moderate to severe effects on oxygen carrying capacity or erythrocyte physiology Hemoglobin; Molecular Motors I

  31. What is a molecular motor? • A protein-based system that interconverts chemical energy and mechanical work • We’ll discuss several molecular motors today, and then next Monday we’ll look at what might be the most important one: the vertebrate muscle. Hemoglobin; Molecular Motors I

  32. Microtubules (G&G §16.3) • 30-nm structures composed of repeating units of a heterodimeric protein, tubulin • -tubulin: 55 kDa • -tubulin: 55 kDa also • Structure of microtubule itself: polymer in which the heterodimers wrap around in a staggered way to produce a tube Hemoglobin; Molecular Motors I

  33. Tubulin structure •  and  are similar but not identical • Structure determined by electron diffraction, not X-ray diffraction • Some NMR structures available too • Two GTP binding sites per monomer • Heterodimer is stable if Ca2+ present Hemoglobin; Molecular Motors I

  34. iClicker quiz question 1 • Why might you expect crystallization of tubulin to be difficult? • (a) It is too big to crystallize • (b) It is too small to crystallize • (c) Proteins that naturally form complex but non-crystalline 3-D structures are resistant to crystallization • (d) It is membrane-bound • (e) none of the above Hemoglobin; Molecular Motors I

  35. Tubulin dimer • G&G Fig. 16.12 Hemoglobin; Molecular Motors I

  36. Microtubule structure G&G fig. 16.12(b) • Polar structure composed of / dimers • Dimers wrap around tube as they move • Asymmetric: growth at plus end Hemoglobin; Molecular Motors I

  37. Treadmilling • Dimers added at plus end while others removed at minus end (GTP-dependent): that effectively moves the microtubule • Fig. 16.13 Hemoglobin; Molecular Motors I

  38. Role in cytoskeleton • Microtubules have a role apart from their role in molecular motor operations: • They are responsible for much of the rigidity of the cytoskeleton • Cytoskeleton contains: • Microtubules (made from tubulin) • Intermediate fibers (7-12nm; made from keratins and other proteins) • Microfilaments (8nm diameter: made from actin) Hemoglobin; Molecular Motors I

  39. Cytoskeletal components • Cf. Fig. 16.11 Hemoglobin; Molecular Motors I

  40. Cilia and flagella • Both are microtubule-based structures used in movement • Cilia: • short, hairlike projections, found on many animal and lower-plant cells • beating motion moves cells or helps move extracellular fluid over surface • Flagella • Longer, found singly or a few at a time • Propel cells through fluids Hemoglobin; Molecular Motors I

  41. Axonemes • Bundle of microtubule fibers: • Two central microtubules • Nine pairs of joined microtubules • Often described as a 9+2 arrangement • Surrounded by plasma membrane that connects to the cell’s PM • If we remove the PM and add a lot of salt, the axoneme will release a protein called dynein Hemoglobin; Molecular Motors I

  42. Axoneme structure • Inner pair connected by bridge • Outer nine pairs connected to each other and to inner pair Hemoglobin; Molecular Motors I

  43. How cilia move • Each outer pair contains asmaller, static A tubule anda larger, dynamic B tubule • Dynein walks along B tubulewhile remaining attached toA tubule of a different pair • Crosslinks mean the axoneme bends • Dynein is a complex protein assembly: • ATPase activity in 2-3 dynein heavy chains • Smaller proteins attach at A-tubule end Hemoglobin; Molecular Motors I

  44. Another view of polymerization M.A.Jordan & L. Wilson (2004), Microtubules as targets for anticancer drugs, Nature Rev. Cancer 4:253 Hemoglobin; Molecular Motors I

  45. Inhibitors of microtubule polymerization • Vinblastine & vincristine are inhibitors: show antitumor activity by shutting down cell division • Colchicine inhibits microtubule polymerization: relieves gout, probably by slowing movement of neutrophils Hemoglobin; Molecular Motors I

  46. Paclitaxel: a stimulator • Formerly called taxol • Stimulates microtubule polymerization • Antitumor activity • Stimulates search for other microtubule polymerization stimulants Hemoglobin; Molecular Motors I

  47. iClicker question 2 1. How do you imagine paclitaxel might work in combating cancer? • (a) by producing frantic cell division • (b) by interfering with microtubule disassembly, preventing cell division • (c) by causing changes in tertiary structures of  and  tubulin • (d) none of the above Hemoglobin; Molecular Motors I

  48. Movement of organelles and vacuoles • Can be fast:2-5 µm s-1 • Hard to study • 1985: Kinesin isolated • 1987: Cytosolic dynein found Hemoglobin; Molecular Motors I

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