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Students: Wouter Spoor, Martijn van Rosmalen, Jim Eijsermans Supervisor: Prof. M. Egmond

Students: Wouter Spoor, Martijn van Rosmalen, Jim Eijsermans Supervisor: Prof. M. Egmond. Global overview protein channel. SecA (motor) and SecYE (channel) Hydrophobic crack Two-helix finger Clamp Plug. SecA. Motor protein T. maritima B. subtilis (PDB) M. jannaschii

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Students: Wouter Spoor, Martijn van Rosmalen, Jim Eijsermans Supervisor: Prof. M. Egmond

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  1. Students: Wouter Spoor, Martijn van Rosmalen, Jim Eijsermans Supervisor: Prof. M. Egmond

  2. Global overview protein channel • SecA (motor) and SecYE (channel) • Hydrophobic crack • Two-helix finger • Clamp • Plug

  3. SecA • Motor protein • T. maritima • B. subtilis (PDB) • M. jannaschii • ATP binding domain (NBD1 & 2) • Polypeptide crosslinking domain (PPXD)

  4. SecA - Topview (cytoplasmic) • Fig A. X-ray of SecA • Fig B. X-ray of SecA with ADP

  5. SecA - Clamp • Hydrophobic residues show where polypeptide can bind Blue shows high conserved regions, red the lowest Orange: conserved hydrophobic residues

  6. SecY • Red: SecA/Fab binding domain • Blue: used for intramolecular crosslinking • Green: used for intermolecular crosslinking

  7. SecYE – Hydrophobic crack • Binding to SecYE results in opening

  8. SecYE – Hydrophobic crack due to conformational change • V329 and T92 cannot form disulfide bonds after conformational change: • Fig F. Addition of SecA results in no disulfide bond between V329C and T92C

  9. SecYE – MD analysis • MD analysis show closing of hydrophobic crack

  10. SecYE – Comparison of M. jannaschii and T. maritima • Pore ring of SecYE opens by conformational change of TM8 & 9 • Plug opens also by this conformational change

  11. SecYE – Hydrophobic residues in Pore ring (cytoplasmic-/topview)

  12. Remember SecYE and SecA • How do SecYE and SecA fit together? SecYE (sideview) SecA (topview)

  13. Alignment of the clamp of SecA and the lateral gate of SecY Red line: signal polypeptide Yellow/blue: SecA Red/green/gray: SecYEG

  14. Conclusion • ATP necessary for closing of clamp • This to hold polypeptide in place • Clamp SecA positioned above lateral gate SecYE • Channel which goes through SecA and SecYE • Hydrophobic crack occurs in SecYE where the two-finger helix is positioned • Two-finger helix “pushes” polypeptide through hydrophobic crack

  15. Model 1: the dimer model • Results brought in consideration: • Polypeptide “holded” in clamp of SecA • SecA positioned central above SecYE • No evidence for dimer formation of SecYE • Suggestion for different model

  16. Model 2: based on results

  17. Discussion • Fab antibody • How much comparable with SecA • Some other crystals as proof • X-ray structure is not representative for dynamics • Visualize interactions between SecA and SecYE

  18. Stability of the SecA-SecY complex

  19. Grey: ADP • Blue: ADP-BeFx

  20. Fab and SecA bind at the same residues

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