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Is ubiquitination always the result of mistakes?

Is ubiquitination always the result of mistakes?. The N-end Rule: The N-terminal amino acid determines half-life. Destabilizing N-termini are recognized by a special E2/E3. The eukaryotic cell cycle is controlled by the ubiquitin pathway.

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Is ubiquitination always the result of mistakes?

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  1. Is ubiquitination always the result of mistakes?

  2. The N-end Rule: The N-terminal amino acid determines half-life Destabilizing N-termini are recognized by a special E2/E3

  3. The eukaryotic cell cycle is controlled by the ubiquitin pathway

  4. During the cells cycle synthesis or mitosis DNA damage signals cell cycle arrest. The Mdm2 E2/E3 keeps p53 abundance low under normal conditions. After DNA damage p53 is stabilized and it causes the trancription of a CDK inhibitor, thereby stopping the cell cycle.

  5. Let’s examine a real world example: The globins

  6. Oxygen Carriers Hemerythrin Hemocyanin Globins

  7. Need metals to bind to oxygen…..why? Oxygen is a diradical It has 2 unpaired electrons 1/23O2 + 1X ---> 1XO

  8. This is the oxygen paradox The spin restriction limits the chemical reactivity by imposing a kinetic barrier Singlet oxygen in the excited state is extraordinarily reactive This is the basis for photodynamic therapy

  9. Metals cause oxygen to become reactive because they are radicals themselves. They eliminate spin restrictions Fe(II)-O2 Fe(III)-O2- Fe(III)-O2- + Fe(II) Fe(III)-O22--Fe(III) Highly reactive! 2Fe(IV)=O Fe(III)-O22--Fe(III) 2Fe(IV)=O Fe(III)-O-Fe(III)

  10. A picket-fence Fe(II)–porphyrin complex with bound O2- Metals, along with proteins, can harness the reactivity of oxygen by activating it an shielding it

  11. Fe(II) binds dioxygen Fe(III) does not Why? Oxygen to metal charge transfer Fe(II)-O2 Fe(III)-O2- Stable Fe(III)-O2 Fe(IV)-O2- Unstable Fe(II) will also bind NO, CO, S2- , CN-

  12. The visible absorption spectra of oxygenated and deoxygenated hemoglobins.

  13. Distal Proximal

  14. C-terminus N-terminus

  15. Fractional saturation of myoglobin with oxygen

  16. Hemoglobin binds oxygen cooperatively This means that the binding of one oxygen to one subunit affects the binding to another subunit

  17. The two state model of hemoglobin binding Oxy or R state Deoxy or T state

  18. Major Structural differences upon oxidation of hemoglobin Fe moves from 0.55Å out of the heme plane to 0.22Å out of the plane Extensive a1-b1 contacts unchanged Minimal a1-b2 contact altered by as much as 6 Å 15º offcenter rotation of the protomers

  19. High spin Oh Fe2+ Low spin Oh Fe3+ x2-y2 x2-y2 z2 z2 xz yz xz yz xy xy Decreased radius Increased radius

  20. Ion pairs that stabilize the T-state 1) Intra-subunitHis-Asp pair 2)  Lys--C-terminus pair 3) Inter- subunit Arg-Asp/C-terminus-Lys pairs 4) Inter- subunit N-terminus-C-terminus pair

  21. High CO2 in tissues decreases the pH: the Bohr effect Low pH stabilizes the T state. How? CO2 + H2O ---> H+ + HCO3-

  22.  Lys--C-terminus pair Intra-subunitHis-Asp pair At low pH His146 is protonated allowing the ion pair to form

  23. R-NH2 + CO2 R-NH-COO- + H+ -amino terminus Carbaminohemoglobin COO- Inter- subunit Arg-Asp/C-terminus-Lys pairs

  24. deoxyHb can also bind chloride ion tightly Cl- is higher in veins than in arteries High Cl- will cause O2 release Inter- subunit Arg-Asp/C-terminus-Lys pairs

  25. Thus the T state is stabilized by: Low pH High CO2 High Cl-

  26. Comparison of the O2-dissociation curves of “stripped” Hb and whole blood in 0.01M NaCl at pH 7.0.

  27. 2,3-bisphosphoglycerate binds deoxyHb BPG Keeps Hb deoxygenated

  28. Binding of BPG to deoxyHb.

  29. The effect of high-altitude exposure on the p50 and the BPG concentration of blood in sea level–adapted individuals.

  30. Notice: 8 mM BPG results in less saturation at high altitude….but….results in equivalent release of O2. Note 38% release of O2 at sea level with 5 mM BPG and 30% release at high altitude with 5 mM BPG. Also note 37% release at high altitude with 8 mM BPG!

  31. Fetal hemoglobin (a2g2) Neonatal hemoglobin (z2e2) Adult hemoglobin (a2b2) 1% adult hemoglobin (a2d2) Why are there different globins?

  32. Myoglobin has a higher affinity for O2 in tissues

  33. Fetal hemoglobin (a2g2) No affinity for BPG Thus it will look more like myoglobin

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