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Is ubiquitination always the result of mistakes?.

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Presentation on theme: "Is ubiquitination always the result of mistakes?."— Presentation transcript:

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

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5 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.

6 Let’s examine a real world example: The globins

7 Oxygen Carriers Hemerythrin Hemocyanin Globins

8 Need metals to bind to oxygen…..why? Oxygen is a diradical It has 2 unpaired electrons 1/2 3 O 2 + 1 X ---> 1 XO

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

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

11 A picket-fence Fe(II)–porphyrin complex with bound O 2 - Metals, along with proteins, can harness the reactivity of oxygen by activating it an shielding it

12 Fe(II) binds dioxygen Fe(III) does not Why? Oxygen to metal charge transfer Fe(II)-O 2 Fe(III)-O 2 - Fe(II) will also bind NO, CO, S 2-, CN - Fe(III)-O 2 Fe(IV)-O 2 - Stable Unstable

13 The visible absorption spectra of oxygenated and deoxygenated hemoglobins.

14 Distal Proximal

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16 N-terminus C-terminus

17 Fractional saturation of myoglobin with oxygen

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

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

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21 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  1-  1 contacts unchanged Minimal  1-  2 contact altered by as much as 6 Å 15º offcenter rotation of the protomers

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24 High spin O h Fe 2+ xy xzyz x 2 -y 2 z2z2 Increased radius Low spin O h Fe 3+ xy xzyz x 2 -y 2 z2z2 Decreased radius

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

26 Low pH stabilizes the T state. How? High CO 2 in tissues decreases the pH: the Bohr effect CO 2 + H 2 O ---> H + + HCO 3 -

27  Lys-  -C-terminus pair Intra-  subunit His-Asp pair At low pH His 146 is protonated allowing the ion pair to form

28 COO- R-NH 2 + CO 2 R-NH-COO - + H + Carbaminohemoglobin  -amino terminus Inter-  subunit Arg-Asp/C-terminus-Lys pairs

29 deoxyHb can also bind chloride ion tightly High Cl - will cause O 2 release Cl - is higher in veins than in arteries Inter-  subunit Arg-Asp/C-terminus-Lys pairs

30 Thus the T state is stabilized by: Low pH High CO 2 High Cl -

31 Comparison of the O 2 -dissociation curves of “stripped” Hb and whole blood in 0.01M NaCl at pH 7.0.

32 2,3-bisphosphoglycerate binds deoxyHb BPG Keeps Hb deoxygenated

33 Binding of BPG to deoxyHb.

34 The effect of high-altitude exposure on the p 50 and the BPG concentration of blood in sea level– adapted individuals.

35 Notice: 8 mM BPG results in less saturation at high altitude….but….results in equivalent release of O 2. Note 38% release of O 2 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!

36 Fetal hemoglobin (  2  2 ) Adult hemoglobin (  2  2 ) Neonatal hemoglobin (  2  2 ) 1% adult hemoglobin (  2  2 ) Why are there different globins?

37 Myoglobin has a higher affinity for O 2 in tissues

38 Fetal hemoglobin (  2  2 ) No affinity for BPG Thus it will look more like myoglobin


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