New methodologies of investigation of model peptides-lipids systems and application to the study of the antimicrobial and transfection peptide LAH4

1. New methodologies of Solid-State NMR and biophysical studies of antimicrobial and designed peptides in model and natural membranes Barbara Perrone Laboratoire de Biophysique et RMN des M´embranes Universit´e de Strasbourg, Strasbourg, France September 13th, 2011 Thesis defense

2. Outline 1 Introduction Motivations 2 Solid State NMR (SS-NMR) Solid-state NMR and Magic Angle Hole problem Magic Angle Hole and Transient Oscillation Holes origins 3 A strategy to reﬁll the Magic Angle Hole and Transient Oscillation Holes Changing the shape of the contact pulse 4 Another strategy: ROtor Directed Exchange of Orientation (RODEO) RODEO - Theory and method development RODEO - Applications 5 Biophysical studies of the antimicrobial peptide LAH4 LAH4-membrane insertion in presence of citrate 6 Future perspective Barbara Perrone (UdS) 13th September 2011 Thesis defense 2 / 55

4. Motivations Antimicrobial Resistance threat to public health Antimicrobial Peptides Solid-state NMR 2011 E.coli outbreak 46 deaths, 3000 persons infected, $2,840,000,000 Mechanisms SS-NMR methodology Barbara Perrone (UdS) 13th September 2011 Thesis defense 4 / 55

6. Solid-state NMR - Anisotropy CSA tensor σPAF =   σ11 0 0 0 σ22 0 0 0 σ33   15 N-labeled amide in a helical peptide σ33 ∼ 200 ppm σ22 ∼ 85 ppm σ11 ∼ 65 ppm Barbara Perrone (UdS) 13th September 2011 Thesis defense 6 / 55

9. Solid-state NMR - Anisotropy CSA tensor 300 200 100 0 ppm !11 !22 !33 σPAF =   σ11 0 0 0 σ22 0 0 0 σ33   15 N-labeled amide in a helical peptide σ33 ∼ 200 ppm σ22 ∼ 85 ppm σ11 ∼ 65 ppm Barbara Perrone (UdS) 13th September 2011 Thesis defense 6 / 55

10. Oriented SS-NMR Mechanically Oriented Samples B0 200 ppm ca B0 80 ppm ca Drawbacks Low coil ﬁlling-factor due to support Problematic environmental control Not suitable for complex membrane or in cell studies Barbara Perrone (UdS) 13th September 2011 Thesis defense 7 / 55

14. Unoriented SS-NMR Fast uniaxial rotational diﬀusion around the bilayer normal Figure: Prongidi-Fix et al., J. Am. Chem. Soc., 2007 15N−KALP in unoriented POPC, 310 K 300 200 100 0 ppm MAH Distortion at the isotropic frequency =“Magic Angle Hole”(MAH) Major problems with line-shape ﬁtting Barbara Perrone (UdS) 13th September 2011 Thesis defense 8 / 55

15. Origins of MAH Cross-Polarization (CP) Magnetization transfer: 1H −→13 C,15 N Dipolar coupling constant: b = −γI γS 2r3 (3 cos2 θ − 1) b(θ∗) = 0 θ∗ = 54.7° Magic Angle Chemical Shift Anisotropya: ∆σ ∝ (3 cos2 θ − 1) σ(θ∗) = σiso a hypothesis: symmetric chemical shift tensor σ parallel to the dipolar vector Barbara Perrone (UdS) 13th September 2011 Thesis defense 9 / 55

16. Static CP under fast uniaxial motion Static CP of ferrocene 150 100 50 ppm τcp = 50 µs Magic Angle Hole (MAH) at the isotropic frequency Transient Oscillation Holes (TOHs) At long contact times, a quasi-equilibrium state is reached, and the powder pattern line-shape is recovered; too long to be used in biological samples (short T1ρ) Barbara Perrone (UdS) 13th September 2011 Thesis defense 10 / 55

20. Static CP under fast uniaxial motion Static CP of ferrocene 150 100 50 ppm τcp = 1 ms Magic Angle Hole (MAH) at the isotropic frequency Transient Oscillation Holes (TOHs) At long contact times, a quasi-equilibrium state is reached, and the powder pattern line-shape is recovered; too long to be used in biological samples (short T1ρ) Barbara Perrone (UdS) 13th September 2011 Thesis defense 10 / 55

23. Origin of the Transient Oscillation Holes (TOHs) Classical ”I-S”model MBKE I-I*-S model ferrocene M¨uller et al., Phys. Rev. Lett., 1974 Figures adapted from Kolodziejski et al., Chem.Rev., 2002 Barbara Perrone (UdS) 13th September 2011 Thesis defense 11 / 55

25. Changing the shape of the contact pulse Shaped-pulse CP Conclusions tCP =50 µs: MAH tCP =150-350 µs: MAH TOHs + 30%S/N tCP =1-3 ms: MAH Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55

26. Changing the shape of the contact pulse Shaped-pulse CP Conclusions tCP =50 µs: MAH tCP =150-350 µs: MAH TOHs + 30%S/N tCP =1-3 ms: MAH Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55

27. Changing the shape of the contact pulse Shaped-pulse CP Conclusions tCP =50 µs: MAH tCP =150-350 µs: MAH TOHs + 30%S/N tCP =1-3 ms: MAH 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) ramp Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55

28. Changing the shape of the contact pulse Shaped-pulse CP Conclusions tCP =50 µs: MAH tCP =150-350 µs: MAH TOHs + 30%S/N tCP =1-3 ms: MAH 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) s45 Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55

31. Changing the shape of the contact pulse Shaped-pulse CP Conclusions tCP =50 µs: MAH tCP =150-350 µs: MAH TOHs + 30%S/N tCP =1-3 ms: MAH 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) s84.3 Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55

35. Changing the shape of the contact pulse Shaped-pulse CP Conclusions tCP =50 µs: MAH tCP =150-350 µs: MAH TOHs + 30%S/N tCP =1-3 ms: MAH 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) rectangular Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55

36. Changing the shape of the contact pulse Shaped-pulse CP Conclusions tCP =50 µs: MAH tCP =150-350 µs: MAH TOHs + 30%S/N tCP =1-3 ms: MAH 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) rectangular 100 50 ppm Figure: s75 CP, tCP = 50 µs 100 50 ppm Figure: rectangular CP, tCP = 50 µs Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55

37. Changing the shape of the contact pulse Shaped-pulse CP Conclusions tCP =50 µs: MAH tCP =150-350 µs: MAH TOHs + 30%S/N tCP =1-3 ms: MAH 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) rectangular 100 50 ppm Figure: s88 CP, tCP = 150 µs 100 50 ppm Figure: rectangular CP, tCP = 150 µs Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55

38. Changing the shape of the contact pulse Shaped-pulse CP Conclusions tCP =50 µs: MAH tCP =150-350 µs: MAH TOHs + 30%S/N tCP =1-3 ms: MAH 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) rectangular 100 50 ppm Figure: s75 CP, tCP = 3 ms 100 50 ppm Figure: rectangular CP, tCP = 3 ms Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55

40. ROtor Directed Exchange of Orientation (RODEO) RODEO-CP pulse sequence Cross-Polarization RODEO Hahn’s echo Acquisition ! ! # !!# # Barbara Perrone (UdS) 13th September 2011 Thesis defense 15 / 55

45. MAT(Magic Angle Turning) provide the orientation-exchange Orientation of the MA cone before and after the mixing time Figure: before tmix Barbara Perrone (UdS) 13th September 2011 Thesis defense 16 / 55

46. MAT(Magic Angle Turning) provide the orientation-exchange Orientation of the MA cone before and after the mixing time Figure: after tmix Barbara Perrone (UdS) 13th September 2011 Thesis defense 16 / 55

47. MAT(Magic Angle Turning) provide the orientation-exchange Orientation of the MA cone before and after the mixing time Figure: intersection (no exchange) Barbara Perrone (UdS) 13th September 2011 Thesis defense 16 / 55

48. RODEO-Theory RODEO Signal: G(t) = Sz(tCP) · · exp iδω0 2ωr sin2 β 2 [sin 2(γ + ωr (t + τm)) − sin 2(γ + ωr τm)] − √ 2 sin 2β [sin(γ + ωr (t + τm)) − sin(γ + ωr τm)] MBKE Solutionab: Sz(t) = 1 − 1 2 exp(−Rdf t) − 1 2 exp − Rdf + Rdp 2 t cos(bt) ϕ = ωr τm between the evolution (CP) and detection (CS) frequencies a M¨uller, Kumar, and Baumann, and Ernst (M¨uller et al., Phys. Rev. Lett., 1974) b δ=CSA, ω0 =Larmor freq., r=angle between rIS and B0, ωr /2π =spinning freq., β=angle between r and the spinning axis, γ=azimuth of r about the spinning axis, Barbara Perrone (UdS) 13th September 2011 Thesis defense 17 / 55

49. RODEO-CP: eﬀect of τm RODEO-CP, MAT @ 55 Hz, τcp = 150 µs As long as τm = nTr nN, RODEO reﬁll the MAH and TOH In black, experimental spectra. In red, theoretical powder-pattern. −20180 160 140 120 100 80 60 40 20 0 ppm−20180 160 140 120 100 80 60 40 20 0 ppm Figure: τm = Tr Barbara Perrone (UdS) 13th September 2011 Thesis defense 18 / 55

50. RODEO-CP: eﬀect of τm RODEO-CP, MAT @ 55 Hz, τcp = 150 µs As long as τm = nTr nN, RODEO reﬁll the MAH and TOH In black, experimental spectra. In red, theoretical powder-pattern. −20180 160 140 120 100 80 60 40 20 0 ppm−20180 160 140 120 100 80 60 40 20 0 ppm Figure: τm = 0.1 Tr Barbara Perrone (UdS) 13th September 2011 Thesis defense 18 / 55

59. RODEO-CP: effect of tCP RODEO-CP, τmix = Tr /2, MAT @ 50 Hz In black the experimental spectra, in red the theoretical fit. 150 100 50 0 ppm150 100 50 0 ppm Figure: τcp = 50µs RODEO-CP removes distortions −→ line-shape fitting −→ δii Barbara Perrone (UdS) 13th September 2011 Thesis defense 19 / 55

62. RODEO-CP: effect of tCP RODEO-CP, τmix = Tr /2, MAT @ 50 Hz In black the experimental spectra, in red the theoretical fit. 150 100 50 0 ppm150 100 50 0 ppm Figure: τcp = 1 ms RODEO-CP removes distortions −→ line-shape fitting −→ δii Barbara Perrone (UdS) 13th September 2011 Thesis defense 19 / 55

63. Spin diffusion contribution Static RODEO-CP, τcp = 50 µs. 150 100 50 0 ppm Figure: τm = 1s ! ! # !! Spin diffusion in ferrocene is not sufficient to refill the MAH. Barbara Perrone (UdS) 13th September 2011 Thesis defense 20 / 55

64. Spin diffusion contribution Static RODEO-CP, τcp = 50 µs. 150 100 50 0 ppm Figure: τm=5 s ! ! # !! Spin diffusion in ferrocene is not sufficient to refill the MAH. Barbara Perrone (UdS) 13th September 2011 Thesis defense 20 / 55

65. Spin diffusion contribution Static RODEO-CP, τcp = 50 µs. 150 100 50 0 ppm Figure: τm=10 s ! ! # !! Spin diffusion in ferrocene is not sufficient to refill the MAH. Barbara Perrone (UdS) 13th September 2011 Thesis defense 20 / 55

66. Magic Angle Turning contribution CP, MAT@50Hz In black, CP turning at the magic angle (50Hz) static CP, τcp =10 ms −20180 160 140 120 100 80 60 40 20 0 ppm Figure: τcp =50 µs ! ! # !!! Slow MAT CP is not suﬃcient to reﬁll the MAH for tCP 1 ms Barbara Perrone (UdS) 13th September 2011 Thesis defense 21 / 55

67. Magic Angle Turning contribution CP, MAT@50Hz In black, CP turning at the magic angle (50Hz) static CP, τcp =10 ms −20180 160 140 120 100 80 60 40 20 0 ppm Figure: τcp =150 µs, ! ! # !!! Slow MAT CP is not suﬃcient to reﬁll the MAH for tCP 1 ms Barbara Perrone (UdS) 13th September 2011 Thesis defense 21 / 55

68. Magic Angle Turning contribution CP, MAT@50Hz In black, CP turning at the magic angle (50Hz) static CP, τcp =10 ms −20180 160 140 120 100 80 60 40 20 0 ppm Figure: τcp =350 µs ! ! # !!! Slow MAT CP is not suﬃcient to reﬁll the MAH for tCP 1 ms Barbara Perrone (UdS) 13th September 2011 Thesis defense 21 / 55

69. Magic Angle Turning contribution CP, MAT@50Hz In black, CP turning at the magic angle (50Hz) static CP, τcp =10 ms −20180 160 140 120 100 80 60 40 20 0 ppm Figure: τcp =1 ms ! ! # !!! Slow MAT CP is not suﬃcient to reﬁll the MAH for tCP 1 ms Barbara Perrone (UdS) 13th September 2011 Thesis defense 21 / 55

70. ∼400Hz - MAT RODEO-CP Spinning faster: MAT @414 Hz 100 80 60 40 20 ppm CP, τcp =150 µs, MAT @ 414 Hz RODEO (MAS@400Hz) improve the line-shape ﬁtting −→ better resolution in structural parameters Barbara Perrone (UdS) 13th September 2011 Thesis defense 22 / 55

71. ∼400Hz - MAT RODEO-CP Spinning faster: MAT @414 Hz 100 80 60 40 20 ppm RODEO-CP, τcp =150 µs, τm = 0.5Tr , MAS @ 414 Hz RODEO (MAS@400Hz) improve the line-shape ﬁtting −→ better resolution in structural parameters Barbara Perrone (UdS) 13th September 2011 Thesis defense 22 / 55

72. ∼400Hz - MAT RODEO-CP Spinning faster: MAT @414 Hz 100 80 60 40 20 ppm100 80 60 40 20 ppm Fit of RODEO-CP, τcp =150 µs, τm = 0.5Tr , MAS @ 414 Hz RODEO (MAS@400Hz) improve the line-shape ﬁtting −→ better resolution in structural parameters Barbara Perrone (UdS) 13th September 2011 Thesis defense 22 / 55

73. Conclusions RODEO RODEO recover the powder pattern line-shape by de-correlating the evolution and detection frequencies by slow turning at the magic angle Simple and robust Suppress MAH and TOHs for contact times longer ≥ 150 µs Even for very short contact times, RODEO spectra line-shape are very close to the theoretical line-shape −→ tensor parameters extracted with good accuracy Overall a loss of 10% in intensity respect to CP due to π/2-pulse imperfections To increase S/N, adiabatic CP and higher MAS (or other angles) can be used. Barbara Perrone (UdS) 13th September 2011 Thesis defense 23 / 55

79. RODEO-CP applied to designed peptides in unoriented model membranes Designed Peptides KL14 in plane KKLLKKAKKLLKK-CONH2 KALP transmembrane GKKLALALALALALALALALKKA-CONH2 Model Membrane POPC 1-palmitoyl-2-oleoyl-phosphatidylcholine Barbara Perrone (UdS) 13th September 2011 Thesis defense 24 / 55

82. RODEO-CP applied to designed peptides in unoriented model membranes σ11, σ22, σ33 σ, σ⊥ Model ! ! # $ % %'' %(( ) 350 300 250 200 150 100 50 0 ppm 350 300 250 200 150 100 50 0 ppm KL14 KALP σ33 (ppm) 228.2±0.5 221±4 σ22 (ppm) 78±4 77.5±0.3 σ11 (ppm) 54±1 55.0±0.2 Barbara Perrone (UdS) 13th September 2011 Thesis defense 25 / 55

83. RODEO-CP applied to designed peptides in unoriented model membranes σ11, σ22, σ33 σ, σ⊥ Model ! ! # $ % %'' %(( ) −50250 200 150 100 50 0 ppm−50250 200 150 100 50 0 ppm −50250 200 150 100 50 0 ppm−50250 200 150 100 50 0 ppm RODEO-APHH-CP, 50 Hz MAT, τcp = 800 µs, P/L=2/100, 298 K KL14 KALP σ (ppm) 72±4 205±4 σ⊥ (ppm) 143.5±0.5 78.7±0.3 Barbara Perrone (UdS) 13th September 2011 Thesis defense 25 / 55

84. RODEO-CP applied to designed peptides in unoriented model membranes σ11, σ22, σ33 σ, σ⊥ Model ! ! # $ % %'' %(( ) σ = σ11cos2αsin2β + σ22sin2αsin2β + σ33cos2β C.Sizun and B.Bechinger, J. Am. Chem. Soc. (2002) 0 Π 2 Π 3 Π 2 2 Π Α 0 Π 2 Π 3 Π 2 2 ΠΒ 100 150 200 Σ −→ α = pitch angle and β= helix tilt (approx: σ33 helix axis, fast rotational diﬀusion around ˆn ) Barbara Perrone (UdS) 13th September 2011 Thesis defense 25 / 55

85. Helix tilt calculation Graphical solution KL14: intersection of the surface σ⊥ = f (α, β) with the experimental plane σ⊥ = 143.5 ppm. 0 Π 4 Π 2 3 Π 2 2 Π Α 0 Π 4 Π 2 3 Π 2 2 Π Β 75 100 125 150 Σ ppm Barbara Perrone (UdS) 13th September 2011 Thesis defense 26 / 55

86. Helix tilt calculation Graphical solution KALP: intersection of the surface σ = f (α, β) with the experimental plane σ = 205 ppm. 0Π 4Π 2 3 Π 2 2 Π Α 0 Π 4 Π 2 3 Π 2 2 Π Β 100 150 200 Σ Barbara Perrone (UdS) 13th September 2011 Thesis defense 26 / 55

87. Results KALP topologically open curve β = f (α). α [0, 2π] β [22.7 − 24.5]° KL14 topologically closed curve β = f (α). α [−63.3, +63.3]° β [70.5, 109.5]° Π 2 ΠΠ 3 Π 2 2 Π Α Π 2 Π 2 Β Barbara Perrone (UdS) 13th September 2011 Thesis defense 27 / 55

88. Results KALP topologically open curve β = f (α). α [0, 2π] β [22.7 − 24.5]° KL14 topologically closed curve β = f (α). α [−63.3, +63.3]° β [70.5, 109.5]° Π 2 ΠΠ 3 Π 2 2 Π Α Π 2 Π 2 Β Π 2 ΠΠ 3 Π 2 2 Π Α Π 2 Π 2 Β Barbara Perrone (UdS) 13th September 2011 Thesis defense 27 / 55

89. RODEO-CP applied to PLAH4 in E.coli lipid extract PLAH4 in E.coli total lipid extract 15N fully labelled (also lateral chains) MLV of E.coli-extracted lipids (P/L=2%) 10 mM tris buﬀer (pH∼ 5) RODEO-APHH-CP, turning at 69 Hz, rotor axis at 80° respective to B0, τm = (0.5Tr ± 18%), τcp = 800 µs, 310K. No line-shape distortions. 250 200 150 100 50 0 ppm Barbara Perrone (UdS) 13th September 2011 Thesis defense 28 / 55

90. RODEO-CP applied to PLAH4 in E.coli lipid extract PLAH4 in E.coli total lipid extract 15N fully labelled (also lateral chains) MLV of E.coli-extracted lipids (P/L=2%) 10 mM tris buﬀer (pH∼ 5) RODEO-APHH-CP, turning at 69 Hz, rotor axis at 80° respective to B0, τm = (0.5Tr ± 18%), τcp = 800 µs, 310K. In red, line-shape ﬁtting. 250 200 150 100 50 0 ppm250 200 150 100 50 0 ppm Barbara Perrone (UdS) 13th September 2011 Thesis defense 28 / 55

91. RODEO-CP applied to PLAH4 in E.coli lipid extract PLAH4 in E.coli total lipid extract 15N fully labelled (also lateral chains) MLV of E.coli-extracted lipids (P/L=2%) 10 mM tris buﬀer (pH∼ 5) RODEO-APHH-CP, turning at 69 Hz, rotor axis at 80° respective to B0, τm = (0.5Tr ± 18%), τcp = 800 µs, 310K. In blue, tensor components: σ = 78 ppm, σ⊥ = 142 ppm. Corresponding estimated values (in-plane): σ = 58−81 ppm, σ⊥ = 142 − 153 ppm. 250 200 150 100 50 0 ppm250 200 150 100 50 0 ppm Barbara Perrone (UdS) 13th September 2011 Thesis defense 28 / 55

92. RODEO-CP applied to PLAH4 in E.coli lipid extract PLAH4 in E.coli total lipid extract 15N fully labelled (also lateral chains) MLV of E.coli-extracted lipids (P/L=2%) 10 mM tris buﬀer (pH∼ 5) RODEO-APHH-CP, turning at 69 Hz, rotor axis at 80° respective to B0, τm = (0.5Tr ± 18%), τcp = 800 µs, 310K. In violet, isotropic components σ ≈ 15N σbackbone iso 250 200 150 100 50 0 ppm250 200 150 100 50 0 ppm250 200 150 100 50 0 ppm250 200 150 100 50 0 ppm Barbara Perrone (UdS) 13th September 2011 Thesis defense 28 / 55

93. RODEO-CP applied to PLAH4 in E.coli lipid extract PLAH4 in E.coli total lipid extract 15N fully labelled (also lateral chains) MLV of E.coli-extracted lipids (P/L=2%) 10 mM tris buﬀer (pH∼ 5) RODEO-APHH-CP, turning at 69 Hz, rotor axis at 80° respective to B0, τm = (0.5Tr ± 18%), τcp = 800 µs, 310K. Assignment of the additional peaks. 250 200 150 100 50 0 ppm250 200 150 100 50 0 ppm250 200 150 100 50 0 ppm250 200 150 100 50 0 ppm H lateral chains K lateral chains lipids (?) Barbara Perrone (UdS) 13th September 2011 Thesis defense 28 / 55

94. RODEO-CP applied to PLAH4 in-vivo E.coli PLAH4 in-vivo E.coli ≤0.75mg 15N fully labeled PLAH4 ∼300 mg bacteria pellet TRIS buﬀer (pH∼7) no nutrients, no O2 RODEO-APHH-CP, 53 Hz MAT, τm = (0.5Tr ± 18%), τCP = 800 µs, 4 days acquisition, 298 K. viability tests: no diﬀerence w or w/o peptide 20% bacteria died S/N can be improved 250 200 150 100 50 0 ppm Barbara Perrone (UdS) 13th September 2011 Thesis defense 29 / 55

96. LAH4 Known properties KKALLALALHHL AHLALHLALALKKA-NH2 a unstructured in solution helical in membrane/micelles a Bechinger(1996), Aisenbrey et al. (1996),Vogt et al.(1999), Kichler et al.(2003), Mason et al. (2006), Kichler et al.(2007), Prongide-Fix et al. (2007), Marquette et al. (2008) pH∼5 protonation of histidines surface-associated pH∼7 deprotonation of histidines transmembrane Barbara Perrone (UdS) 13th September 2011 Thesis defense 31 / 55

97. LAH4 in presence of citrate buffer Oriented Solid-State NMR 15N single labeled LAH4 in oriented DMPC (P/L=1:50) DMPC= 1,2-dimyristoyl-sn-glycero-3-phosphocholine No buffer, pH ∼5a 200 100 0 ppm Figure: σ ≈80 ppm =⇒In-plane orientation. a With 10 mM citrate buffer, pH 5 200 100 0 ppm Figure: σ ≈ 200 ppm =⇒ Transmembrane orientation. Barbara Perrone (UdS) 13th September 2011 Thesis defense 32 / 55

98. LAH4 in presence of citrate buﬀer-I Oriented Circular Dichroism absence of a negative band around 208 nm is an indication of a TM helix Barbara Perrone (UdS) 13th September 2011 Thesis defense 33 / 55

99. LAH4 in presence of citrate buﬀer-I Oriented Circular Dichroism absence of a negative band around 208 nm is an indication of a TM helix Barbara Perrone (UdS) 13th September 2011 Thesis defense 33 / 55

100. Small Angle X-ray Scattering (SAXS) Membrane hydrophobic thickness Effect of LAH4 on the hydrophobic thickness of POPC, POPG and POPC/POPG vesicles in citrate buffer pH=5 Bilayer thickness: dB = 2(zH + 2σH) Membrane hydrophobic thickness: dCC = dB − 10˚A ͗f͑q͒͘ϭF͑q͒ϭ2F ͑q͒ϩF ͑q͒, ͑7͒ intensity is therefore given by the diffraction of the phospho- lipid multilayers within the quasi-long-range order lattice, plus the additional diffuse scattering of single, uncorrelated bilayers I͑q͒ϰ 1 q2 „͉F͑q͉͒2 S͑q͒ϩNdiff͉F͑q͉͒2 …. ͑13͒ In further context of this paper we will refer to the above described model as MCG, since it is a combination of MCT and a Gaussian electron density representation of the head- group ͓30͔. A further benefit of this method is that one can derive structural parameters from simple geometric relationships, without the need of volumetric data as, e.g., in the approach of McIntosh and Simon ͓32͔, or Nagle et al. ͓14͔. For deter- FIG. 1. Electron density profile model ␳(z) as a function of distance z from the center of the bilayer, given by a summation of two Gaussians ͓see Eq. ͑5͔͒. 4002 PRE 62PABST, RAPPOLT, AMENITSCH, AND LAGGNER Barbara Perrone (UdS) 13th September 2011 Thesis defense 34 / 55

101. Small Angle X-ray Scattering (SAXS) Membrane hydrophobic thickness Effect of LAH4 on the hydrophobic thickness of POPC, POPG and POPC/POPG vesicles in citrate buffer pH=5 Bilayer thickness: dB = 2(zH + 2σH) Membrane hydrophobic thickness: dCC = dB − 10˚A ͗f͑q͒͘ϭF͑q͒ϭ2F ͑q͒ϩF ͑q͒, ͑7͒ intensity is therefore given by the diffraction of the phospho- lipid multilayers within the quasi-long-range order lattice, plus the additional diffuse scattering of single, uncorrelated bilayers I͑q͒ϰ 1 q2 „͉F͑q͉͒2 S͑q͒ϩNdiff͉F͑q͉͒2 …. ͑13͒ In further context of this paper we will refer to the above described model as MCG, since it is a combination of MCT and a Gaussian electron density representation of the head- group ͓30͔. A further benefit of this method is that one can derive structural parameters from simple geometric relationships, without the need of volumetric data as, e.g., in the approach of McIntosh and Simon ͓32͔, or Nagle et al. ͓14͔. For deter- FIG. 1. Electron density profile model ␳(z) as a function of distance z from the center of the bilayer, given by a summation of two Gaussians ͓see Eq. ͑5͔͒. 4002 PRE 62PABST, RAPPOLT, AMENITSCH, AND LAGGNER Barbara Perrone (UdS) 13th September 2011 Thesis defense 34 / 55

102. Conclusions Conclusion LAH4 in citrate inserts in a transmembrane manner in DMPC, even at acidic pH, when histidines are charged. LAH4 assume assumes an in-plane alignment in DMPC when no buﬀer is added, in agreement with previous results in other lipids (POPC). The membrane thickening POPC at pH 5 in the presence of citrate buﬀer, suggest that the peptide inserts in a transmembrane manner. Barbara Perrone (UdS) 13th September 2011 Thesis defense 35 / 55

106. Future perspective RODEO-Applications-LAH4 Improve in vivo E.coli RODEO experiment Compare results obtained in E.coli lipid LAH4 and citrate: open questions peculiar behavior of the citrate anion or is it general? what is the mechanism? does it aﬀect the antimicrobial activity? Barbara Perrone (UdS) 13th September 2011 Thesis defense 37 / 55

111. Acknowledgments Thanks to: Prof. Dr. B.Bechinger Prof. Dr. B.Wallace Dr. C. Marques Prof. Dr. Willumeit Prof. Dr. N. C. Nielsen Dr. J.Raya Dr. J.Hirschinger Dr. E.Glattard Dr. V.Vidovic Dr. A.Miles Prof. Dr. K.Lohner Dr. G.Pabst Laboratory of NMR and Biophysics of Membranes Biocontrol Network EU FP6 Funding Barbara Perrone (UdS) 13th September 2011 Thesis defense 38 / 55

112. Shaped-pulse CP experiments - 50 µs CP on ferrocene powder - SetA ramp CP, tCP = 50 µs rectangular CP, tCP = 10 ms. 100 50 ppm 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) CP on ferrocene powder - SetB Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55

113. Shaped-pulse CP experiments - 50 µs CP on ferrocene powder - SetA s45a CP, tCP = 50 µs rectangular CP performed at tCP = 10 ms. 100 50 ppm 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) a “sφ”tangent-amplitude shapes built on the formula ω1I (t) − ω1S (t) = dIS tan φ(τ 2 − t) (Hediger et al., 1994) Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55

114. Shaped-pulse CP experiments - 50 µs CP on ferrocene powder - SetA s65 CP, tCP = 50 µs rectangular CP performed at tCP = 10 ms. 100 50 ppm 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) CP on ferrocene powder - SetB Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55

115. Shaped-pulse CP experiments - 50 µs CP on ferrocene powder - SetA s75 CP, tCP = 50 µs rectangular CP, tCP = 10 ms. 100 50 ppm 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) CP on ferrocene powder - SetB Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55

116. Shaped-pulse CP experiments - 50 µs CP on ferrocene powder - SetA s84.3 CP, tCP = 50 µs rectangular CP, tCP = 10 ms. 100 50 ppm 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) CP on ferrocene powder - SetB Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55

117. Shaped-pulse CP experiments - 50 µs CP on ferrocene powder - SetA s88 CP, tCP = 50 µs rectangular CP, tCP = 10 ms. 100 50 ppm 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) CP on ferrocene powder - SetB Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55

120. Shaped-pulse CP experiments - 50 µs CP on ferrocene powder - SetA rectangular CP, tCP = 50 µs rectangular CP, tCP = 10 ms. 100 50 ppm 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) CP on ferrocene powder - SetB Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55

121. Shaped-pulse CP experiments - 50 µs CP on ferrocene powder - SetA CP on ferrocene powder - SetB ramp CP, tCP = 50 µs rectangular CP, tCP = 10 ms. 100 50 ppm 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55

130. Shape variations on static CP experiments - 150 µs 1H −13 C CP on ferrocene powder performed with tCP = 150 µs applying the shaped-pulse shown below on 13C (SetA). Confront with rectangular CP performed at tCP = 10 ms. 100 50 ppm 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55

131. Shape variations on static CP experiments - 150 µs 1H −13 C CP on ferrocene powder performed with tCP = 150 µs applying the shaped-pulse shown below on 13C (SetA). Confront with rectangular CP performed at tCP = 10 ms. 100 50 ppm100 50 ppm 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55

132. Shape variations on static CP experiments - 150 µs 1H −13 C CP on ferrocene powder performed with tCP = 150 µs applying the shaped-pulse shown below on 13C (SetA). Confront with rectangular CP performed at tCP = 10 ms. 100 50 ppm100 50 ppm 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55

139. Shape variations on static CP experiments - 150 µs 1H −13 C CP on ferrocene powder performed with tCP = 150 µs applying the shaped-pulse shown below on 13C (SetB). Confront with rectangular CP performed at tCP = 10 ms. 100 50 ppm 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55

166. Shape variations on static CP experiments - 1 ms 1H −13 C CP on ferrocene powder performed with tCP = 1 ms applying the shaped-pulse shown below on 13C (SetA). Confront with rectangular CP performed at tCP = 10 ms. 100 50 ppm 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55

175. Shape variations on static CP experiments - 1 ms 1H −13 C CP on ferrocene powder performed with tCP = 1 ms applying the shaped-pulse shown below on 13C (SetB). Confront with rectangular CP performed at tCP = 10 ms. 100 50 ppm 0,0 1,0 Contact Time (arbitrary units) 0 20 40 60 80 100 13Ccontactfield(kHz) Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55

202. Introduction to CP - how? Homonuclear spin couple I-I Conservative“Flip-Flop” transitions Heteronuclear spin couple I-S Transitions NOT conservative Double rotating frame with ωRF I = ωRF S Hartmann-Hahn condition: γI ωI = γS ωS Barbara Perrone (UdS) 13th September 2011 Thesis defense 44 / 55

206. RODEO-CP: τm optimization Experimental Figure: τm = Tr 2 Calculated RODEO-CP: µs, MAT@55Hz;CP with tcp = 10 ms. Random-sampling τm results in a RODEO-CP spectra closer to the quasi-equilibrium line-shape. Barbara Perrone (UdS) 13th September 2011 Thesis defense 45 / 55

207. CP dynamics Classical I-S model Thermodynamic approach I(t) follows a double exponential law ferrocene does not follow this law (M¨uller et al., 1974) MBKE I-I*-S model Barbara Perrone (UdS) 13th September 2011 Thesis defense 46 / 55

210. CP dynamics Classical I-S model MBKE I-I*-S model Network of coupled I nuclei Transient harmonic oscillations Figures from Kolodziejski et al., Chem.Rev., 2002 Barbara Perrone (UdS) 13th September 2011 Thesis defense 46 / 55

211. CP dynamics Classical I-S model MBKE I-I*-S model Network of coupled I nuclei Transient harmonic oscillations Figures from Kolodziejski et al., Chem.Rev., 2002 Barbara Perrone (UdS) 13th September 2011 Thesis defense 46 / 55

212. MKBE model MKBE Solution Master equation: ˙σ(t) = −i [H(t), σ(t)] − Γ [σ(t), σ(∞)] Γ = Rdf ([Ix [Ix , σ]] + [Iy [Iy , σ]]) + Rdp [Iz [Iz, σ]] MKBE Solutionab: Sz(t) = 1 − 1 2 exp(−Rdf t) − 1 2 exp − Rdf + Rdp 2 t cos(bt) damped oscillations: freq. depends on b and decay depends on Rdp , Rdf the approach to the ﬁnal equilibrium is regulated by Rdf a M¨uller, Kumar, and Baumann, and Ernst. b |ω1I | = |ω1S |, ω0i ≈ ωRFi ,H(t) = H, b = −γI γS 2r3 IS (3 cos2 θ − 1), T1ρ = 0, |ω1I | + |ω1S | b Rdp, Rdf Barbara Perrone (UdS) 13th September 2011 Thesis defense 47 / 55

216. Oriented SS-NMR Mechanically Oriented Samples B0 200 ppm ca B0 80 ppm ca The projection of the tensor on the axis parallel to B0, σzz, gives a direct indication of the σ33 orientation Θ: σzz = σ11sin2 Θcos2 Φ + σ22sin2 Θsin2 Φ + σ33cos2 Θ ∼ 200 ppm ←→ TRANSMEMBRANE ∼ 80 ppm ←→ IN-PLANE Drawbacks Oriented samples challenging to obtain Problematic environmental control Low ﬁlling factor of the coil Barbara Perrone (UdS) 13th September 2011 Thesis defense 48 / 55

220. Helix tilt calculation Graphical solution σ = σ11cos2αsin2β + σ22sin2αsin2β + σ33cos2β σ⊥ = σ11(1−cos2αsin2β)+σ22(1−sin2αsin2β)+σ33sin2β 2 KL14: intersection of the surfaces σ,⊥ = f (α, β) with the experimental values, i.e. the planes σ = 72.1 ppm and σ⊥ = 143.5 ppm. 0 Π 4 Π 2 Π 3 Π 2 2 Π Α 0Π 4Π 2 Π 3 Π 2 2 Π Β 100 150 200 Σ 0 Π 4 Π 2 3 Π 2 2 Π Α 0 Π 4 Π 2 3 Π 2 2 Π Β 75 100 125 150 Σ ppm Barbara Perrone (UdS) 13th September 2011 Thesis defense 49 / 55

221. Helix tilt calculation Graphical solution σ = σ11cos2αsin2β + σ22sin2αsin2β + σ33cos2β σ⊥ = σ11(1−cos2αsin2β)+σ22(1−sin2αsin2β)+σ33sin2β 2 KALP: intersection of the surfaces σ,⊥ = f (α, β) with the experimental values, i.e. the planes σ=205 ppm and σ⊥ = 78.7 ppm. 0 Π Π 2 3 Π 2 2 Π Α 0 Π Π 2 3 Π 2 2 Π Β 100 150 Σ 0Π 4Π 2 3 Π 2 2 Π Α 0 Π 4 Π 2 3 Π 2 2 Π Β 100 150 200 Σ Barbara Perrone (UdS) 13th September 2011 Thesis defense 49 / 55

222. SAXS data - POPC POPC Figure: Diﬀraction patterns of POPC vesicles with increasing amount of LAH4. Barbara Perrone (UdS) 13th September 2011 Thesis defense 50 / 55

223. POPC Figure: Diﬀraction patterns of POPC vesicles with increasing amount of LAH4. Barbara Perrone (UdS) 13th September 2011 Thesis defense 51 / 55

224. POPG Figure: Diﬀraction patterns of POPG vesicles with increasing amount of LAH4. Barbara Perrone (UdS) 13th September 2011 Thesis defense 52 / 55

225. POPC/POPG 3:1 Figure: Diﬀraction patterns of POPC vesicles with increasing amount of LAH4. Barbara Perrone (UdS) 13th September 2011 Thesis defense 53 / 55

226. Electron Density Proﬁles Electron Density Proﬁles - POPC Barbara Perrone (UdS) 13th September 2011 Thesis defense 54 / 55

227. Electron Density Proﬁles Electron Density Proﬁles - POPG Barbara Perrone (UdS) 13th September 2011 Thesis defense 54 / 55

228. Electron Density Proﬁles Electron Density Proﬁles - POPC/POPG Barbara Perrone (UdS) 13th September 2011 Thesis defense 54 / 55

229. DLS and ﬂuorescence quencing Barbara Perrone (UdS) 13th September 2011 Thesis defense 55 / 55

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