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Quiz_20.09.04

Quiz_20.09.04. 1235-1245 h. Quiz_20.09.04.

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Quiz_20.09.04

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  1. Quiz_20.09.04 1235-1245 h

  2. Quiz_20.09.04 • β lactam antibiotics cross barriers of the bacterial cell to reach their targets. Draw two diagrams cell envelopes: one for G-negative cells, the other for G-positive cells. Label each diagram fully by identifying all macromolecules found in every layer. [5 points] • Show the pathway for traverse of a β lactam across the two cell walls. List the cell’s barriers against antibiotic activity. [3 points] • Inside a Living Cell: coloured diagrams to be submitted [2 points]

  3. Bacterial transport MFS Group translocation ABC transporters OM receptors

  4. MFS • Symport • 2 solutes simultaneously • Same direction across CM • Anion + H+ • Sugar + H+  • LacY of E. coli

  5. Antiport • 2 solutes simultaneously • Opposite directions • NhaA of E. coli • Active uniport • Flow of one ion • Driven directly by ion gradient: K+ • KcsA of E. coli

  6. LacY • lacY of E. coli 46.5 kDa • Integral CM protein • 12 transmembrane helices • Reconsitution • LacY purified in detergent • + P-lipids  • sonicate  • membrane vesicles with LacY

  7. LacY • LacY in vesicles • Add 14C-lactose + H+ • Measurements • 14C inside vesicles • medium: ↓ [H+] ; more basic • Conclusion: • single protein acts as symporter • H.R. Kaback: Science August 1, 2003

  8. Group translocation • Accumulate sugars as P~esters • ~P from PEP • PTS = PEP:carbohydrate phosphotransferase system • Multiple genes • Shared functions • Unique functions

  9. PTS • Sugar1: requires A + B + C  sugar1~P • Sugar2: requires A + B + D  sugar2~P • Sugar3: requires A + B + E  sugar3~P • A and B • Soluble proteins • Cytoplasm • C, D, E • CM proteins

  10. PTS_soluble proteins • enzyme I + PEP  enzyme I~P + pyruvate • E. coli: ptsI locus • 2 identical polypeptides; 58 kDa; dimer • 10,000 copies / cell • His~P • enzyme I~P + HPr  HPr~P + enzyme I • E. coli: ptsH locus • monomer • 100,000 copies / cell

  11. PTS_membrane proteins • HPr~P + enzyme II  HPr + enzyme II~P • enzyme II • Sugar-specific • E II mannitol; also glucitol • E II glucose; also glucosamine, 2-deoxyglucose • E II mannose; also fructose • Fine structure mapping • Binding of sugar • Interaction of HPr~P • Site for transphosphorylation

  12. PTS_membrane proteins • Mechanism of enzyme II • “pore” normally closed • Activated by HPr~P • “pore” opens • P. Maloney: • electron crystallography of enzyme II

  13. ABC transporters • Binding protein-dependent systems • Expt: • cold osmotic shock: 4 °C, sucrose • plasmolysis of E. coli: CM retracts • periplasmic contents: shocked outside cell • 100s soluble proteins: concentrate = shockate • 1. test cell for transport: his, mal • 2. test shockate……..

  14. ABC transporters • shockate • Lyophilize 100s of proteins • Suspend in 1 ml; add to dialysis bag • Buffer, 500 ml • + 14C-histidine • or + 14C-maltose • Proteins in dialysis bag bind radiolabel • Concln: periplasmic contents contain BPs

  15. ABC transporters • Molecular genetics • Maltose transport • malA locus: 74 min; catabolism • malB locus: 91 min; transport • Promoter: malP • malE-malF-malG • MalE = maltose BP (MBP), periplasm • MalF + MalG = CM complex

  16. Promoter: malP • malK-lamB • MalK = ATP hydrolysis, cytoplasmic face of CM • LamB = maltoporin, OM

  17. Maltose transport: mechanism • OM: maltose + LamB • LamB’s greasy slide • Periplasm: maltose finds MBP • MBP: open, bilobar • MBP + maltose  MBP closes; maltose in cleft • Venus fly trap • CM: [MBP+maltose] finds MalF-MalG • Maltose transferred to MalF-MalG cmplx • MalK2: hydrolysis of ATP

  18. OM receptors • Scavenge scarce nutrients • Iron-containing cmpds = siderophores • Regulated by iron supply • Vitamin B12 • IROMPs • FhuA, FepA, Cir, FecA, FhuE • BtuB

  19. OM receptors • Mechanism • Binding of siderophore to IROMP • Signalling to other proteins in periplasm • Movement of siderophore across receptor • Transfer to periplasmic BP • Energy requiring: TonB-ExbB-ExbD

  20. Ferrichrome transport in E. coli ferrichrome OM FhuA TonB CM

  21. OM receptors • Structures of 3 IROMPs • Purify protein • Grow protein crystals • X-ray crystallography  3D of polypeptide chain

  22. OM receptor FhuA: 2 domains • N terminus: cork • C terminus: • βbarrel, 22 strands; • Surface-exposed loops • Short connecting turns: periplasm • Signalling: • Minus siderophore = α helix • Plus siderophore = helix  random coil • Tells energy transducing system to activate

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