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The "pH-A c tivated Trigger" Mechanism of Colicin E1 Channel Domain

The "pH-A c tivated Trigger" Mechanism of Colicin E1 Channel Domain. Abdi Musse MSc. Final Examination. Supervisor Dr. A. R . Merrill. Advisory committee Dr. G. Harauz Dr. F. J. Sharom. Outline. Introduction Research Objectives Results and Discussion Summary and Conclusions.

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The "pH-A c tivated Trigger" Mechanism of Colicin E1 Channel Domain

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  1. The "pH-Activated Trigger" Mechanism of Colicin E1 Channel Domain Abdi Musse MSc. Final Examination Supervisor Dr. A. R. Merrill Advisory committee Dr. G. Harauz Dr. F. J. Sharom

  2. Outline • Introduction • Research Objectives • Results and Discussion • Summary and Conclusions

  3. Overview • The Biology of Pore-forming Colicins • Antimicrobial proteins that are secreted by Escherichia coli • Targets the cytoplasmic membrane • Forms lethaly depolarizing ion channels • Dissipations of the cationic gradients (H+, K+, Na+) Colicin E1

  4. T R C H2N COOH R T C Colicin Ia Wiener et al. (1997) Structure and Function Tol Network (TolC, A and Q) BtuB Receptor Channel-forming

  5. Elkins et al. (1997) The Channel Domain H4 H6 H7 H5 H8 H9 H3 H1 H10 H2 • 2.5 Å Structure of P190 • Three- layered sandwich structure

  6. Interactions with Membranes Activated-intermediate Membrane-anchored Precursor

  7.  Formations of the Open Channel The precursor Voltage-gated • The open channel • Monomer • 4 – 9 Ådiameter

  8. Mechanism of Activation • Acid-induced activation is common to most toxins • Onset of Protein unfolding • Increased structural flexibility • Potentiates the massive unfloding events requisite for membrane insertion and channel formation

  9. H4 H5a Merrill et al. (1997) The pH-Activated Trigger Hypothesis • The trigger motif: helices 4 and 5a • Activating helix-to-coil transition of the trigger motif • Disruption of the critical H-bonds formed by D-408, D-410 and D-423

  10. The Research Objectives • Purpose • To test the proposed pH-activated trigger mechanism • Approaches • Replacements of the critical acidic residues with serine • Incorporation of a disulphide bond within the trigger motif • Tools • Membrane binding • Insertion kinetics • Channel activity • Structural elucidations

  11. Mutant Proteins of Colicin E1 • Asp  Ser • D410S • D408S • D408S/D410S D408S/D423S D410S/D423S • D408S/D410S/D423S • Ala  Cys • A407C/A411C • Single Trp • F413W • F413W/D408S/D423S

  12. Cytotoxicity

  13. WT (folded) WT (7 M GnHCl) Structural Integrity

  14. Probing Free Sulfahydral Side-chains in A407C/A411C with MIANS Non-fluorescent

  15. Presence of a Disulfide Bond in A407C/A411C Channel Peptide MIANS fluorescence Stoichiometry of MIANS Conjugation WT (GnHCl) A407C/A411C (GnHCl) WT (folded) A407C/A411C (folded)

  16. TNP TNP TNP TNP Fluorescence Quenching Membrane Binding

  17. pKa 4 Typical Binding Profile for the WT Channel Peptide

  18. The Expected profile for the Asp  Ser Mutants

  19. The Expected profile for the Asp  Ser Mutants b

  20. b c The Expected Profile for the Disulphide Bonded Mutant

  21. The Binding Profile for the WT protein • Expected pH-binding profile • The effective pKa 4.1 (0.1)

  22. The Binding Profiles of the Double AspSer Mutants • Alkaline-directed shift in binding profile • Consistent with the predicted profile of an altered trigger mechanism

  23. A407C/A411C The Binding Profiles of the Disulphide Bonded Mutant • Un-expected binding profile • At pH 4.5: Ka = 1.4 (0.2) mM-1 (reduced) • 1.7 (0.3) mM-1 (oxidized)

  24. Fluorescence Quenching Membrane Insertion

  25. Time Course of the Fluorescence Quenching

  26. D410 D408 H-bond Salt bridge Apparent Rates of Membrane Insertion

  27. In vitro Channel Activity Cl- Cl- Cl- Fluorescence Dequenching Efflux

  28. Time Course of the Fluorescence Dequenching

  29. The Initial Rate of Cl- Efflux

  30. W-424 W-413

  31. The Time-resolved and Steady-state Fluorescence of the Single Trp Mutants

  32. Time-resolved and Steady-state Fluorescence Parameters

  33. The Time-resolved and Steady-state Fluorescence Parameters

  34. The Time-resolved and Steady-state Fluorescence Parameters

  35. Time-resolved and Steady-state Fluorescence Parameters

  36. The Trigger Residues

  37. The Topology of the Trigger Motif

  38. Possible Implications for the in vivo Mechanism of Activation H1 H1 Trigger Docking site

  39. Summary and Conclusions • These observations confirm the proposed pH-activated trigger mechanism of colicin E1 • Asp  Ser mutations disrupted criticall H-bonds within the tirgger motif • Elevated binding, insertion, and channel activities at near-neutral pH • Shift in the helix-to-coil transition of the trigger motif toward random Coil-like conformational state for helix 4

  40. Colleagues in the Merrill Laboratory Tanya Brodeur Susan Yates Tania Roberts Gerry Prentice* Dave Teal Zahir Hussein Acknowledgements Advisor Dr. A. R. Merrill • Advisory Committee • Dr. G. Harauz • Dr. F. J. Sharom Examining Committee Dr. G. Harauz Dr. P. D. Josephy Dr. M. Palmer *Special thanks

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