1. Unraveling Helix 2 of Colicin E1

2. Background

3. Zakharov et al., BBA. 2004 family of antimicrobial proteins secreted upon environmental stress (regulated by SOS system) three modes of action: 1. depolarization through ion channels 2. inhibition of protein and peptidoglycan synthesis 3. degradation of nucleic acids 1994 – crystal structures of polypeptide fragments of Col E1 1997 – complete crystal structure of channel-forming P190 fragment

4. Cleavage sites: Zhang and Cramer, 1992 Palmer and Merrill, JBC, 1994 Parallax method of depth dependent fluorescence quenching of trp Y367 classed as moderately buried

5. Tory and Merrill, JBC, 1999 Fluorescence and FRET study bimolecular quench constant Δ λ emission max (W) lifetime red blue reduced accessibility Y367 immersed or interfacial

6. Tory and Merrill, BBA, 2002 Red-edge excitation shift analysis no REES exposed or embedded moderate to large REES interfacial no REES buried Y367 interfacial and sequestered from aqueous solvent

7. Musse et al., sometime soon! Helix one displayed an alpha-helical nature in both soluble and membrane-bound states. No elongation or blending of helices 1 and 2. These results support the toroidal pore model.

8. Models

9. Zakharov and Cramer, BBA, 2002 Zahkarov et al., Biophys. J., 2004 Model for colicin import Zakharov and Cramer, BBA, 2002 Colicin membrane insertion

10. Zakharov and Cramer, BBA, 2002 Zakhorov et al., BBA, 2004 Shai, BBA, 1999 Models for membrane insertion

11. My Job

12. Helix 2 of Colicin E1 E365 K366 Y367 S368 K369 M370 A371 Q372 E373 L374 A 375 D376 K377 S378 K379 G380 hydrophobic polar acidic basic

13. Mutagenesis, Expression, Purification and Labelling

14. S378 G380 P190H MutantYield Y367C4.6 S368C2.5 K369C25 M370C3.0 A371C13 A371C-C47 Q372C7.0 E373C4.4 L374C1.4 A375C2.5 D376C8.0 K377C2.6 K377C-C34 S378C11.4 K379C23 G380C28 P190H20 C505A50

15. mBBr  well characterized  relatively small (about the size of a tryptophan)  uncharged, non-perturbing (structure or binding)  essentially non fluorescent until conjugated  fluorescence quenched by near-by W and, to a lesser extent, Y

16. Mutant% labelling Y367C110 S368C100 K369C85 M370C56 A371C9 A371C-C9 Q372C82 E373C90 L374C95 A375C97 D376C84 K377C200 K377C-C83 S378C60 K379C73 G380C81 Bimane Labelling Efficiency M370C - C5057.23 Å A371C – C50510.98 Å E373C - C5056.95 Å L374C - C5057.21 Å K377C - C5058.32 Å

17. Analysis

18. Intrinsic Trp Fluorescence  indication of folded integrity of mutant protein compared with WT  using 295 nm excitation avoids fluorescence from the 9 Y residues  the three trp residues are in rigid environments and exhibit limited flexibility

19. Trp λ emission maximum MutantNativeLabelled Y367C333332 S368C326 K369C326 M370C329325 A371C325324 A371C-C324 Q372C325327 E373C327 L374C332331 A375C326324 D376C326325 K377C328NA K377C-C324325 S378C326 K379C323324 G380C325 P190H324NA C505A324NA

20. SPQ in vitro Channel Assay  test of the pore-forming ability of the mutant protein protein TX-100

21. SPQ Mutant% P190H Rate Y367C9 S368C102 K369C74 M370C82 A371C-C46 Q372C57 E373C63 L374C79 A375C162 D376C126 K377C-C67 S378C105 K379C92 G380C68 C505A67

22. Bimane Fluorescence  reports on accessibility of bimane probe which relates to “location” within the tertiary structure of the protein Musse and Merrill standard apparent polarity scale curve bimane-N-acetyl-Cys dioxane-water solvent system

23. Bimane λ emission maximum MutantSolubleLUV Y367C470469 S368C474471 K369C480470 M370C469466 A371CNA A371C-C455457 Q372C472 E373C473471 L374C468470 A375C464 D376C473471 K377C-C482469 S378C471460 K379C481476 G380C479474

24. Surface Area Solvent Accessibility GETAREA 1.1 Solvent Accessible Surface Areas, Atomic Solvation Energies, and Their Gradients for Macromolecules Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX 77555 Area Per residue Job identifier: get_a_13306 Probe radius : 1.400 Residue Total Apolar Backbone Sidechain Ratio(%) In/Out ILE 345 121.48 109.19 13.62 107.86 73.2 o LYS 346 151.45 27.52 15.63 135.82 82.6 o ASP 347 102.95 34.60 0.13 102.82 91.0 o ALA 348 6.96 6.68 3.62 3.34 5.1 i VAL 349 74.04 74.04 0.00 74.04 60.5 o ASP 350 89.67 30.09 8.52 81.15 71.8 o ALA 351 36.79 35.19 7.92 28.87 44.5 THR 352 0.04 0.04 0.00 0.04 0.0 i VAL 353 89.36 89.33 0.13 89.23 73.0 o SER 354 68.44 41.93 7.49 60.95 78.7 o

25. SASA and bimane λ em max MutantTotal Areaλ em max Y367C1.35470 S368C2.99474 K369C126.82480 M370C6.14469 A371C-C0.76455 Q372C60.79472 E373C80.86473 L374C0.98468 A375C0.00464 D376C70.03473 K377C-C95.47482 S378C0.00471 K379C87.14481 G380C67.90479

26. Anisotropy  predicts local environment within tertiary structure by measuring rotational property of bimane moiety on cysteine residue low value = free moving high value = restricted movement

27. Probe mobility (1/r) MutantSolubleLUV Y367C5.214.65 S368C5.925.85 K369C9.437.41 M370C10.534.93 A371C9.266.21 A371C-C7.415.15 Q372C5.215.15 E373C7.876.54 L374C6.215.49 A375C5.785.41 D376C8.706.85 K377C-C10.646.85 S378C6.374.83 K379C9.718.20 G380C10.537.87 >30% 10-25% <5%

28. Quantum Yield  = number of photons emitted/the number of photons absorbed = fraction of fluorophore that decays through emission low value = accessible high value = not accessible

29. Quantum Yield MutantΔQFΔQF relative Q F Y367C0.1541.312 S368C0.1491.882 K369C-0.0100.979 M370C-0.0070.973 A371CNA A371C-C0.0391.438 Q372C0.0351.156 E373C0.0231.057 L374C0.0021.010 A375C0.2782.390 D376C0.0051.011 K377C-C0.1471.241 S378C0.3461.779 K379C0.0151.039 G380C0.0741.147 >30% 10-25% <5%

30. Dual Quenching Assay Erwin London  depth study  quenching with KI (shallow) and 10-DN (deep)  quenching is insensitive to variation in lipid content  10-DN retains “free energy” over range of bilayer depth  could do study with each quencher alone, but ratio between shallow and deep increases sensitivity and eliminates non-depth related quenching effects (excited state lifetime of bimane)  ratio is linearly dependent on depth of trp residue

31. low ratio value = accessible high ratio value = buried DQA Mutant (Fo/F 10-DN -1)/(Fo/F KI -1) Y367C0.10 S368C0.07 K369C0.03 M370C0.51 A371C-C0.48 Q372C0.05 E373C0.18 L374C0.19 A375C0.70 D376C0.10 K377C-C0.04 S378C0.34 K379C0.40 G380C0.34 >0.4 0.11-0.4 <0.1

32. Summary Bimane lambda emission max (membrane-bound vs soluble): Blue shifted (455-460 nm): A371, S378 Intermediate (461-470 nm): Y367, K369, M370, L374, A375, K377 Red shifted (>471 nm): S368, Q372, E373, D376, K379, G380 Soluble anisotropy: Accessible (0.095-0.135): K369, M370, A371, E373, D376, K377, K379, G380 Inaccessible (0.157-0.192): Y367, S368, Q372, L374, A375, S378 These residues are considered buried, but mBBr may be mobile within “pocket” of protein structure. These residues are considered exposed, but probe may be immobilized by tertiary structure contacts. Membrane bound anisotropy: Little change (<5%): Y367, S368, Q372, A375 Moderate change (10-25%): K369, E373, L374, D376, S378, K379, G380 Significant change (>30%): M370, A371, K377 Quantum Yield: Little change (<5%): K369, M370, E373, L374, D376, K379 Moderate change (10-25%): Y367, Q372, K377, G380 Significant change (>30%): S368, A371, A375, S378 S368 and A371 had rather low soluble Q F values (<0.18, the observed value for Cys-bimane standard) which may have been caused by quenching of the bimane signal by Y367. Dual Quenching Analysis: Accessible (<0.10): Y367, S368, K369, Q372, D376, K377 In-between (0.11-0.4): E373, L374, S378, K379, G380 Buried (>0.4): M370, A371, A375

33. Sobko et al, FEBS Lett, 2004