New methodologies of Solid-State NMR and biophysicalstudies of antimicrobial and designed peptides in modeland natural mem...
Outline1 IntroductionMotivations2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and...
Outline1 IntroductionMotivations2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and...
MotivationsAntimicrobial Resistancethreat to public healthAntimicrobial PeptidesSolid-state NMR2011 E.coli outbreak46 deat...
Outline1 IntroductionMotivations2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and...
Solid-state NMR - AnisotropyCSA tensorσPAF =σ11 0 00 σ22 00 0 σ3315N-labeled amide in a helical peptideσ33 ∼ 200 ppmσ2...
Solid-state NMR - AnisotropyCSA tensorσPAF =σ11 0 00 σ22 00 0 σ3315N-labeled amide in a helical peptideσ33 ∼ 200 ppmσ2...
Solid-state NMR - AnisotropyCSA tensorσPAF =σ11 0 00 σ22 00 0 σ3315N-labeled amide in a helical peptideσ33 ∼ 200 ppmσ2...
Solid-state NMR - AnisotropyCSA tensor300 200 100 0 ppm!11!22!33σPAF =σ11 0 00 σ22 00 0 σ3315N-labeled amide in a heli...
Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caDrawbacksLow coilfilling-factor due tosupportProblematic...
Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caDrawbacksLow coilfilling-factor due tosupportProblematic...
Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caDrawbacksLow coilfilling-factor due tosupportProblematic...
Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caDrawbacksLow coilfilling-factor due tosupportProblematic...
Unoriented SS-NMRFast uniaxial rotational diffusion around the bilayer normalFigure: Prongidi-Fix et al., J. Am. Chem. Soc....
Origins of MAHCross-Polarization (CP)Magnetization transfer: 1H −→13 C,15 NDipolar coupling constant:b = −γI γS 2r3 (3 cos...
Static CP under fast uniaxial motionStatic CP of ferrocene150 100 50 ppmτcp = 50 µsMagic Angle Hole (MAH)at the isotropic ...
Static CP under fast uniaxial motionStatic CP of ferrocene150 100 50 ppmτcp = 50 µsMagic Angle Hole (MAH)at the isotropic ...
Static CP under fast uniaxial motionStatic CP of ferrocene150 100 50 ppmτcp = 150 µsMagic Angle Hole (MAH)at the isotropic...
Static CP under fast uniaxial motionStatic CP of ferrocene150 100 50 ppmτcp = 350 µsMagic Angle Hole (MAH)at the isotropic...
Static CP under fast uniaxial motionStatic CP of ferrocene150 100 50 ppmτcp = 1 msMagic Angle Hole (MAH)at the isotropic f...
Static CP under fast uniaxial motionStatic CP of ferrocene150 100 50 ppmτcp = 3 msMagic Angle Hole (MAH)at the isotropic f...
Static CP under fast uniaxial motionStatic CP of ferrocene150 100 50 ppmτcp = 10 msMagic Angle Hole (MAH)at the isotropic ...
Origin of the Transient Oscillation Holes (TOHs)Classical ”I-S”model MBKE I-I*-S modelferroceneM¨uller et al., Phys. Rev. ...
Outline1 IntroductionMotivations2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and...
Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-...
Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-...
Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-...
Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-...
Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-...
Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-...
Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-...
Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-...
Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-...
Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-...
Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-...
Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-...
Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-...
Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-...
Outline1 IntroductionMotivations2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and...
ROtor Directed Exchange of Orientation (RODEO)RODEO-CP pulse sequenceCross-PolarizationRODEOHahn’s echoAcquisition!!#!!# #...
ROtor Directed Exchange of Orientation (RODEO)RODEO-CP pulse sequenceCross-PolarizationRODEOHahn’s echoAcquisition!!#!!# #...
ROtor Directed Exchange of Orientation (RODEO)RODEO-CP pulse sequenceCross-PolarizationRODEOHahn’s echoAcquisition!!#!!# #...
ROtor Directed Exchange of Orientation (RODEO)RODEO-CP pulse sequenceCross-PolarizationRODEOHahn’s echoAcquisition!!#!!# #...
ROtor Directed Exchange of Orientation (RODEO)RODEO-CP pulse sequenceCross-PolarizationRODEOHahn’s echoAcquisition!!#!!# #...
MAT(Magic Angle Turning) provide theorientation-exchangeOrientation of the MA cone before and after the mixing timeFigure:...
MAT(Magic Angle Turning) provide theorientation-exchangeOrientation of the MA cone before and after the mixing timeFigure:...
MAT(Magic Angle Turning) provide theorientation-exchangeOrientation of the MA cone before and after the mixing timeFigure:...
RODEO-TheoryRODEO Signal:G(t) = Sz(tCP)  ·· expiδω02ωrsin2β2 [sin 2(γ + ωr (t + τm)) − sin 2(γ + ωr τm)]−√2 sin 2β [sin(γ ...
RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn black, exper...
RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn black, exper...
RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn black, exper...
RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn black, exper...
RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn black, exper...
RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn black, exper...
RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn black, exper...
RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn black, exper...
RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn black, exper...
RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn black, exper...
RODEO-CP: effect of tCPRODEO-CP, τmix = Tr /2, MAT @ 50 HzIn black the experimental spectra, in red the theoretical fit.150 ...
RODEO-CP: effect of tCPRODEO-CP, τmix = Tr /2, MAT @ 50 HzIn black the experimental spectra, in red the theoretical fit.150 ...
RODEO-CP: effect of tCPRODEO-CP, τmix = Tr /2, MAT @ 50 HzIn black the experimental spectra, in red the theoretical fit.150 ...
RODEO-CP: effect of tCPRODEO-CP, τmix = Tr /2, MAT @ 50 HzIn black the experimental spectra, in red the theoretical fit.150 ...
Spin diffusion contributionStatic RODEO-CP, τcp = 50 µs.150 100 50 0 ppmFigure: τm = 1s!!#!!Spin diffusion in ferrocene is n...
Spin diffusion contributionStatic RODEO-CP, τcp = 50 µs.150 100 50 0 ppmFigure: τm=5 s!!#!!Spin diffusion in ferrocene is no...
Spin diffusion contributionStatic RODEO-CP, τcp = 50 µs.150 100 50 0 ppmFigure: τm=10 s!!#!!Spin diffusion in ferrocene is n...
Magic Angle Turning contributionCP, MAT@50HzIn black, CP turning at the magic angle (50Hz)static CP, τcp =10 ms−20180 160 ...
Magic Angle Turning contributionCP, MAT@50HzIn black, CP turning at the magic angle (50Hz)static CP, τcp =10 ms−20180 160 ...
Magic Angle Turning contributionCP, MAT@50HzIn black, CP turning at the magic angle (50Hz)static CP, τcp =10 ms−20180 160 ...
Magic Angle Turning contributionCP, MAT@50HzIn black, CP turning at the magic angle (50Hz)static CP, τcp =10 ms−20180 160 ...
∼400Hz - MAT RODEO-CPSpinning faster: MAT @414 Hz100 80 60 40 20 ppmCP, τcp =150 µs, MAT @ 414 HzRODEO (MAS@400Hz) improve...
∼400Hz - MAT RODEO-CPSpinning faster: MAT @414 Hz100 80 60 40 20 ppmRODEO-CP, τcp =150 µs, τm = 0.5Tr , MAS @ 414 HzRODEO ...
∼400Hz - MAT RODEO-CPSpinning faster: MAT @414 Hz100 80 60 40 20 ppm100 80 60 40 20 ppmFit of RODEO-CP, τcp =150 µs, τm = ...
ConclusionsRODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by sl...
ConclusionsRODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by sl...
ConclusionsRODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by sl...
ConclusionsRODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by sl...
ConclusionsRODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by sl...
ConclusionsRODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by sl...
RODEO-CP applied to designed peptides in unorientedmodel membranesDesigned PeptidesKL14in planeKKLLKKAKKLLKK-CONH2KALPtran...
RODEO-CP applied to designed peptides in unorientedmodel membranesDesigned PeptidesKL14in planeKKLLKKAKKLLKK-CONH2KALPtran...
RODEO-CP applied to designed peptides in unorientedmodel membranesDesigned PeptidesKL14in planeKKLLKKAKKLLKK-CONH2KALPtran...
RODEO-CP applied to designed peptides in unorientedmodel membranesσ11, σ22, σ33σ, σ⊥Model!!# $%%%(()350 300 250 200 150 10...
RODEO-CP applied to designed peptides in unorientedmodel membranesσ11, σ22, σ33σ, σ⊥Model!!# $%%%(()−50250 200 150 100 50 ...
RODEO-CP applied to designed peptides in unorientedmodel membranesσ11, σ22, σ33σ, σ⊥Model!!# $%%%(()σ = σ11cos2αsin2β + σ2...
Helix tilt calculationGraphical solutionKL14: intersection of the surface σ⊥ = f (α, β) with the experimentalplane σ⊥ = 14...
Helix tilt calculationGraphical solutionKALP: intersection of the surface σ = f (α, β) with the experimentalplane σ = 205 ...
ResultsKALPtopologically open curveβ = f (α).α [0, 2π]β [22.7 − 24.5]°KL14topologically closed curveβ = f (α).α [−63.3, +6...
ResultsKALPtopologically open curveβ = f (α).α [0, 2π]β [22.7 − 24.5]°KL14topologically closed curveβ = f (α).α [−63.3, +6...
RODEO-CP applied to PLAH4 in E.coli lipid extractPLAH4 in E.coli total lipid extract15N fully labelled (also lateral chain...
RODEO-CP applied to PLAH4 in E.coli lipid extractPLAH4 in E.coli total lipid extract15N fully labelled (also lateral chain...
RODEO-CP applied to PLAH4 in E.coli lipid extractPLAH4 in E.coli total lipid extract15N fully labelled (also lateral chain...
RODEO-CP applied to PLAH4 in E.coli lipid extractPLAH4 in E.coli total lipid extract15N fully labelled (also lateral chain...
RODEO-CP applied to PLAH4 in E.coli lipid extractPLAH4 in E.coli total lipid extract15N fully labelled (also lateral chain...
RODEO-CP applied to PLAH4 in-vivo E.coliPLAH4 in-vivo E.coli≤0.75mg 15N fully labeledPLAH4∼300 mg bacteria pelletTRIS buffe...
Outline1 IntroductionMotivations2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and...
LAH4Known propertiesKKALLALALHHL AHLALHLALALKKA-NH2aunstructured in solutionhelical in membrane/micellesaBechinger(1996), ...
LAH4 in presence of citrate bufferOriented Solid-State NMR15N single labeled LAH4 in oriented DMPC (P/L=1:50)DMPC= 1,2-dimy...
LAH4 in presence of citrate buffer-IOriented Circular Dichroismabsence of a negative band around 208 nm is an indication of...
LAH4 in presence of citrate buffer-IOriented Circular Dichroismabsence of a negative band around 208 nm is an indication of...
Small Angle X-ray Scattering (SAXS)Membrane hydrophobic thicknessEffect of LAH4 on thehydrophobic thickness ofPOPC, POPG an...
Small Angle X-ray Scattering (SAXS)Membrane hydrophobic thicknessEffect of LAH4 on thehydrophobic thickness ofPOPC, POPG an...
ConclusionsConclusionLAH4 in citrate inserts in a transmembrane manner in DMPC, evenat acidic pH, when histidines are char...
ConclusionsConclusionLAH4 in citrate inserts in a transmembrane manner in DMPC, evenat acidic pH, when histidines are char...
ConclusionsConclusionLAH4 in citrate inserts in a transmembrane manner in DMPC, evenat acidic pH, when histidines are char...
Outline1 IntroductionMotivations2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and...
Future perspectiveRODEO-Applications-LAH4Improve in vivo E.coli RODEO experimentCompare results obtained in E.coli lipidLA...
Future perspectiveRODEO-Applications-LAH4Improve in vivo E.coli RODEO experimentCompare results obtained in E.coli lipidLA...
Future perspectiveRODEO-Applications-LAH4Improve in vivo E.coli RODEO experimentCompare results obtained in E.coli lipidLA...
Future perspectiveRODEO-Applications-LAH4Improve in vivo E.coli RODEO experimentCompare results obtained in E.coli lipidLA...
Future perspectiveRODEO-Applications-LAH4Improve in vivo E.coli RODEO experimentCompare results obtained in E.coli lipidLA...
AcknowledgmentsThanks to:Prof. Dr.B.BechingerProf. Dr.B.WallaceDr. C. MarquesProf. Dr. WillumeitProf. Dr. N. C.NielsenDr. ...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAramp CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAs45a CP, tCP = 50 µsrectangular CP performed at tCP = 10 m...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAs65 CP, tCP = 50 µsrectangular CP performed at tCP = 10 ms...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAs75 CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm ...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAs84.3 CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 pp...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAs88 CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm ...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAs89.5 CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 pp...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAs89.9 CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 pp...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetArectangular CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangul...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangul...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangul...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangul...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangul...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangul...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangul...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangul...
Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangul...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe ...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shap...
Introduction to CP - how?Homonuclear spin couple I-IConservative“Flip-Flop”transitionsHeteronuclear spin couple I-STransit...
Introduction to CP - how?Homonuclear spin couple I-IConservative“Flip-Flop”transitionsHeteronuclear spin couple I-STransit...
Introduction to CP - how?Homonuclear spin couple I-IConservative“Flip-Flop”transitionsHeteronuclear spin couple I-STransit...
Introduction to CP - how?Homonuclear spin couple I-IConservative“Flip-Flop”transitionsHeteronuclear spin couple I-STransit...
RODEO-CP: τm optimizationExperimentalFigure: τm = Tr2CalculatedRODEO-CP: µs, MAT@55Hz;CP with tcp = 10 ms.Random-sampling ...
CP dynamicsClassical I-S modelThermodynamic approachI(t) follows a double exponential lawferrocene does not follow this la...
CP dynamicsClassical I-S modelThermodynamic approachI(t) follows a double exponential lawferrocene does not follow this la...
CP dynamicsClassical I-S modelThermodynamic approachI(t) follows a double exponential lawferrocene does not follow this la...
CP dynamicsClassical I-S modelMBKE I-I*-S modelNetwork of coupled I nucleiTransient harmonic oscillationsFigures from Kolo...
CP dynamicsClassical I-S modelMBKE I-I*-S modelNetwork of coupled I nucleiTransient harmonic oscillationsFigures from Kolo...
MKBE modelMKBE SolutionMaster equation:˙σ(t) = −i [H(t), σ(t)] − Γ [σ(t), σ(∞)]Γ = Rdf ([Ix [Ix , σ]] + [Iy [Iy , σ]]) + R...
MKBE modelMKBE SolutionMaster equation:˙σ(t) = −i [H(t), σ(t)] − Γ [σ(t), σ(∞)]Γ = Rdf ([Ix [Ix , σ]] + [Iy [Iy , σ]]) + R...
MKBE modelMKBE SolutionMaster equation:˙σ(t) = −i [H(t), σ(t)] − Γ [σ(t), σ(∞)]Γ = Rdf ([Ix [Ix , σ]] + [Iy [Iy , σ]]) + R...
MKBE modelMKBE SolutionMaster equation:˙σ(t) = −i [H(t), σ(t)] − Γ [σ(t), σ(∞)]Γ = Rdf ([Ix [Ix , σ]] + [Iy [Iy , σ]]) + R...
Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caThe projection of the tensor on the axisparallel to B0,...
Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caThe projection of the tensor on the axisparallel to B0,...
Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caThe projection of the tensor on the axisparallel to B0,...
Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caThe projection of the tensor on the axisparallel to B0,...
Helix tilt calculationGraphical solutionσ = σ11cos2αsin2β + σ22sin2αsin2β + σ33cos2βσ⊥ = σ11(1−cos2αsin2β)+σ22(1−sin2αsin2...
Helix tilt calculationGraphical solutionσ = σ11cos2αsin2β + σ22sin2αsin2β + σ33cos2βσ⊥ = σ11(1−cos2αsin2β)+σ22(1−sin2αsin2...
SAXS data - POPCPOPCFigure: Diffraction patterns of POPC vesicles with increasing amount of LAH4.Barbara Perrone (UdS) 13th...
POPCFigure: Diffraction patterns of POPC vesicles with increasing amount of LAH4.Barbara Perrone (UdS) 13thSeptember 2011 T...
POPGFigure: Diffraction patterns of POPG vesicles with increasing amount of LAH4.Barbara Perrone (UdS) 13thSeptember 2011 T...
POPC/POPG 3:1Figure: Diffraction patterns of POPC vesicles with increasing amount of LAH4.Barbara Perrone (UdS) 13thSeptemb...
Electron Density ProfilesElectron Density Profiles - POPCBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 54 / 55
Electron Density ProfilesElectron Density Profiles - POPGBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 54 / 55
Electron Density ProfilesElectron Density Profiles - POPC/POPGBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 54 / 55
DLS and fluorescence quencingBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 55 / 55
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New methodologies of investigation of model peptides-lipids systems and application to the study of the antimicrobial and transfection peptide LAH4

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The tilt angle of helical peptides reconstituted in non-oriented membranes can be derived by 15N powder patterns line-shape analysis. This approach allows more physiological-like conditions compared with conventional oriented SS-NMR spectroscopy, but spectral distortions at the isotropic chemical shift compromise the precision and the interpretation of the measure. Our new method, RODEO, recovers the theoretical powder pattern line-shape by ROtor-Directed Exchange of Orientations Cross-Polarization. Firstly, RODEO was tested on designed peptides in unoriented model membranes. Successively, we applied it to the antimicrobial peptide PLAH4 in extracted lipid mixtures, and, for the first time, in vivo, in Escherichia coli.
In the second part of this work, we present some unexpected solid-state NMR, oriented circular dichroism and X-ray scattering results of the antimicrobial peptide LAH4 in the presence of citrate. It was previously shown that the LAH4-helix adopts an in-plane orientation in acidic conditions, while, at neutral pH, the peptide adopts a trans-membrane orientation. In contrast, we found that when citrate buffer is added to regulate the pH at 5, the peptide inserts in a transmembrane manner. Some possible explanations are suggested.

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

  1. 1. New methodologies of Solid-State NMR and biophysicalstudies of antimicrobial and designed peptides in modeland natural membranesBarbara PerroneLaboratoire de Biophysique et RMN des M´embranesUniversit´e de Strasbourg, Strasbourg, FranceSeptember 13th, 2011Thesis defense
  2. 2. Outline1 IntroductionMotivations2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and Transient Oscillation Holes origins3 A strategy to refill the Magic Angle Hole and Transient Oscillation HolesChanging the shape of the contact pulse4 Another strategy: ROtor Directed Exchange of Orientation (RODEO)RODEO - Theory and method developmentRODEO - Applications5 Biophysical studies of the antimicrobial peptide LAH4LAH4-membrane insertion in presence of citrate6 Future perspectiveBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 2 / 55
  3. 3. Outline1 IntroductionMotivations2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and Transient Oscillation Holes origins3 A strategy to refill the Magic Angle Hole and Transient Oscillation HolesChanging the shape of the contact pulse4 Another strategy: ROtor Directed Exchange of Orientation (RODEO)RODEO - Theory and method developmentRODEO - Applications5 Biophysical studies of the antimicrobial peptide LAH4LAH4-membrane insertion in presence of citrate6 Future perspectiveBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 3 / 55
  4. 4. MotivationsAntimicrobial Resistancethreat to public healthAntimicrobial PeptidesSolid-state NMR2011 E.coli outbreak46 deaths, 3000 persons infected,$2,840,000,000MechanismsSS-NMR methodologyBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 4 / 55
  5. 5. Outline1 IntroductionMotivations2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and Transient Oscillation Holes origins3 A strategy to refill the Magic Angle Hole and Transient Oscillation HolesChanging the shape of the contact pulse4 Another strategy: ROtor Directed Exchange of Orientation (RODEO)RODEO - Theory and method developmentRODEO - Applications5 Biophysical studies of the antimicrobial peptide LAH4LAH4-membrane insertion in presence of citrate6 Future perspectiveBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 5 / 55
  6. 6. Solid-state NMR - AnisotropyCSA tensorσPAF =σ11 0 00 σ22 00 0 σ3315N-labeled amide in a helical peptideσ33 ∼ 200 ppmσ22 ∼ 85 ppmσ11 ∼ 65 ppmBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 6 / 55
  7. 7. Solid-state NMR - AnisotropyCSA tensorσPAF =σ11 0 00 σ22 00 0 σ3315N-labeled amide in a helical peptideσ33 ∼ 200 ppmσ22 ∼ 85 ppmσ11 ∼ 65 ppmBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 6 / 55
  8. 8. Solid-state NMR - AnisotropyCSA tensorσPAF =σ11 0 00 σ22 00 0 σ3315N-labeled amide in a helical peptideσ33 ∼ 200 ppmσ22 ∼ 85 ppmσ11 ∼ 65 ppmBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 6 / 55
  9. 9. Solid-state NMR - AnisotropyCSA tensor300 200 100 0 ppm!11!22!33σPAF =σ11 0 00 σ22 00 0 σ3315N-labeled amide in a helical peptideσ33 ∼ 200 ppmσ22 ∼ 85 ppmσ11 ∼ 65 ppmBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 6 / 55
  10. 10. Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caDrawbacksLow coilfilling-factor due tosupportProblematicenvironmentalcontrolNot suitable forcomplex membraneor in cell studiesBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 7 / 55
  11. 11. Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caDrawbacksLow coilfilling-factor due tosupportProblematicenvironmentalcontrolNot suitable forcomplex membraneor in cell studiesBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 7 / 55
  12. 12. Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caDrawbacksLow coilfilling-factor due tosupportProblematicenvironmentalcontrolNot suitable forcomplex membraneor in cell studiesBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 7 / 55
  13. 13. Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caDrawbacksLow coilfilling-factor due tosupportProblematicenvironmentalcontrolNot suitable forcomplex membraneor in cell studiesBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 7 / 55
  14. 14. Unoriented SS-NMRFast uniaxial rotational diffusion around the bilayer normalFigure: Prongidi-Fix et al., J. Am. Chem. Soc., 200715N−KALP inunoriented POPC,310 K300 200 100 0 ppmMAHDistortion at the isotropicfrequency =“Magic AngleHole”(MAH)Major problems withline-shape fittingBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 8 / 55
  15. 15. Origins of MAHCross-Polarization (CP)Magnetization transfer: 1H −→13 C,15 NDipolar coupling constant:b = −γI γS 2r3 (3 cos2 θ − 1) b(θ∗) = 0 θ∗ = 54.7° Magic AngleChemical Shift Anisotropya: ∆σ ∝ (3 cos2 θ − 1) σ(θ∗) = σisoahypothesis: symmetric chemical shift tensor σ parallel to the dipolar vectorBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 9 / 55
  16. 16. Static CP under fast uniaxial motionStatic CP of ferrocene150 100 50 ppmτcp = 50 µsMagic Angle Hole (MAH)at the isotropic frequencyTransient OscillationHoles (TOHs)At long contact times, aquasi-equilibrium state isreached, and the powderpattern line-shape isrecovered; too long to beused in biological samples(short T1ρ)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 10 / 55
  17. 17. Static CP under fast uniaxial motionStatic CP of ferrocene150 100 50 ppmτcp = 50 µsMagic Angle Hole (MAH)at the isotropic frequencyTransient OscillationHoles (TOHs)At long contact times, aquasi-equilibrium state isreached, and the powderpattern line-shape isrecovered; too long to beused in biological samples(short T1ρ)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 10 / 55
  18. 18. Static CP under fast uniaxial motionStatic CP of ferrocene150 100 50 ppmτcp = 150 µsMagic Angle Hole (MAH)at the isotropic frequencyTransient OscillationHoles (TOHs)At long contact times, aquasi-equilibrium state isreached, and the powderpattern line-shape isrecovered; too long to beused in biological samples(short T1ρ)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 10 / 55
  19. 19. Static CP under fast uniaxial motionStatic CP of ferrocene150 100 50 ppmτcp = 350 µsMagic Angle Hole (MAH)at the isotropic frequencyTransient OscillationHoles (TOHs)At long contact times, aquasi-equilibrium state isreached, and the powderpattern line-shape isrecovered; too long to beused in biological samples(short T1ρ)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 10 / 55
  20. 20. Static CP under fast uniaxial motionStatic CP of ferrocene150 100 50 ppmτcp = 1 msMagic Angle Hole (MAH)at the isotropic frequencyTransient OscillationHoles (TOHs)At long contact times, aquasi-equilibrium state isreached, and the powderpattern line-shape isrecovered; too long to beused in biological samples(short T1ρ)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 10 / 55
  21. 21. Static CP under fast uniaxial motionStatic CP of ferrocene150 100 50 ppmτcp = 3 msMagic Angle Hole (MAH)at the isotropic frequencyTransient OscillationHoles (TOHs)At long contact times, aquasi-equilibrium state isreached, and the powderpattern line-shape isrecovered; too long to beused in biological samples(short T1ρ)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 10 / 55
  22. 22. Static CP under fast uniaxial motionStatic CP of ferrocene150 100 50 ppmτcp = 10 msMagic Angle Hole (MAH)at the isotropic frequencyTransient OscillationHoles (TOHs)At long contact times, aquasi-equilibrium state isreached, and the powderpattern line-shape isrecovered; too long to beused in biological samples(short T1ρ)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 10 / 55
  23. 23. Origin of the Transient Oscillation Holes (TOHs)Classical ”I-S”model MBKE I-I*-S modelferroceneM¨uller et al., Phys. Rev. Lett.,1974Figures adapted fromKolodziejski et al., Chem.Rev., 2002Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 11 / 55
  24. 24. Outline1 IntroductionMotivations2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and Transient Oscillation Holes origins3 A strategy to refill the Magic Angle Hole and Transient Oscillation HolesChanging the shape of the contact pulse4 Another strategy: ROtor Directed Exchange of Orientation (RODEO)RODEO - Theory and method developmentRODEO - Applications5 Biophysical studies of the antimicrobial peptide LAH4LAH4-membrane insertion in presence of citrate6 Future perspectiveBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 12 / 55
  25. 25. Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-3 ms: MAHBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 13 / 55
  26. 26. Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-3 ms: MAHBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 13 / 55
  27. 27. Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-3 ms: MAH0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)rampBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 13 / 55
  28. 28. Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-3 ms: MAH0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)s45Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 13 / 55
  29. 29. Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-3 ms: MAH0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)s65Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 13 / 55
  30. 30. Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-3 ms: MAH0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)s75Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 13 / 55
  31. 31. Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-3 ms: MAH0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)s84.3Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 13 / 55
  32. 32. Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-3 ms: MAH0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)s88Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 13 / 55
  33. 33. Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-3 ms: MAH0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)s89.5Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 13 / 55
  34. 34. Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-3 ms: MAH0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)s89.9Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 13 / 55
  35. 35. Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-3 ms: MAH0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)rectangularBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 13 / 55
  36. 36. Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-3 ms: MAH0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)rectangular100 50 ppmFigure: s75 CP, tCP = 50 µs100 50 ppmFigure: rectangular CP, tCP = 50 µsBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 13 / 55
  37. 37. Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-3 ms: MAH0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)rectangular100 50 ppmFigure: s88 CP, tCP = 150 µs100 50 ppmFigure: rectangular CP, tCP = 150 µsBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 13 / 55
  38. 38. Changing the shape of the contact pulseShaped-pulse CP ConclusionstCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/NtCP =1-3 ms: MAH0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)rectangular100 50 ppmFigure: s75 CP, tCP = 3 ms100 50 ppmFigure: rectangular CP, tCP = 3 msBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 13 / 55
  39. 39. Outline1 IntroductionMotivations2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and Transient Oscillation Holes origins3 A strategy to refill the Magic Angle Hole and Transient Oscillation HolesChanging the shape of the contact pulse4 Another strategy: ROtor Directed Exchange of Orientation (RODEO)RODEO - Theory and method developmentRODEO - Applications5 Biophysical studies of the antimicrobial peptide LAH4LAH4-membrane insertion in presence of citrate6 Future perspectiveBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 14 / 55
  40. 40. ROtor Directed Exchange of Orientation (RODEO)RODEO-CP pulse sequenceCross-PolarizationRODEOHahn’s echoAcquisition!!#!!# #Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 15 / 55
  41. 41. ROtor Directed Exchange of Orientation (RODEO)RODEO-CP pulse sequenceCross-PolarizationRODEOHahn’s echoAcquisition!!#!!# #Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 15 / 55
  42. 42. ROtor Directed Exchange of Orientation (RODEO)RODEO-CP pulse sequenceCross-PolarizationRODEOHahn’s echoAcquisition!!#!!# #Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 15 / 55
  43. 43. ROtor Directed Exchange of Orientation (RODEO)RODEO-CP pulse sequenceCross-PolarizationRODEOHahn’s echoAcquisition!!#!!# #Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 15 / 55
  44. 44. ROtor Directed Exchange of Orientation (RODEO)RODEO-CP pulse sequenceCross-PolarizationRODEOHahn’s echoAcquisition!!#!!# #Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 15 / 55
  45. 45. MAT(Magic Angle Turning) provide theorientation-exchangeOrientation of the MA cone before and after the mixing timeFigure: before tmixBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 16 / 55
  46. 46. MAT(Magic Angle Turning) provide theorientation-exchangeOrientation of the MA cone before and after the mixing timeFigure: after tmixBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 16 / 55
  47. 47. MAT(Magic Angle Turning) provide theorientation-exchangeOrientation of the MA cone before and after the mixing timeFigure: intersection (no exchange)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 16 / 55
  48. 48. RODEO-TheoryRODEO Signal:G(t) = Sz(tCP) ·· expiδω02ωrsin2β2 [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 − 12 exp(−Rdf t) − 12 exp−Rdf +Rdp2tcos(bt)ϕ = ωr τm between the evolution (CP) and detection (CS) frequenciesaM¨uller, Kumar, and Baumann, and Ernst (M¨uller et al., Phys. Rev. Lett., 1974)bδ=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) 13thSeptember 2011 Thesis defense 17 / 55
  49. 49. RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn 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 ppmFigure: τm = TrBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 18 / 55
  50. 50. RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn 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 ppmFigure: τm = 0.1 TrBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 18 / 55
  51. 51. RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn 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 ppmFigure: τm = 0.2 TrBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 18 / 55
  52. 52. RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn 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 ppmFigure: τm = 0.3 TrBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 18 / 55
  53. 53. RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn 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 ppmFigure: τm = 0.4 TrBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 18 / 55
  54. 54. RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn 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 ppmFigure: τm = 0.5 TrBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 18 / 55
  55. 55. RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn 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 ppmFigure: τm = 0.6 TrBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 18 / 55
  56. 56. RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn 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 ppmFigure: τm = 0.7 TrBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 18 / 55
  57. 57. RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn 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 ppmFigure: τm = 0.8 TrBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 18 / 55
  58. 58. RODEO-CP: effect of τmRODEO-CP, MAT @ 55 Hz, τcp = 150 µsAs long as τm = nTr nN, RODEO refill the MAH and TOHIn 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 ppmFigure: τm = 0.9 TrBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 18 / 55
  59. 59. RODEO-CP: effect of tCPRODEO-CP, τmix = Tr /2, MAT @ 50 HzIn black the experimental spectra, in red the theoretical fit.150 100 50 0 ppm150 100 50 0 ppmFigure: τcp = 50µsRODEO-CP removes distortions −→ line-shape fitting −→ δiiBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 19 / 55
  60. 60. RODEO-CP: effect of tCPRODEO-CP, τmix = Tr /2, MAT @ 50 HzIn black the experimental spectra, in red the theoretical fit.150 100 50 0 ppm150 100 50 0 ppmFigure: τcp = 150µsRODEO-CP removes distortions −→ line-shape fitting −→ δiiBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 19 / 55
  61. 61. RODEO-CP: effect of tCPRODEO-CP, τmix = Tr /2, MAT @ 50 HzIn black the experimental spectra, in red the theoretical fit.150 100 50 0 ppm150 100 50 0 ppmFigure: τcp = 350µsRODEO-CP removes distortions −→ line-shape fitting −→ δiiBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 19 / 55
  62. 62. RODEO-CP: effect of tCPRODEO-CP, τmix = Tr /2, MAT @ 50 HzIn black the experimental spectra, in red the theoretical fit.150 100 50 0 ppm150 100 50 0 ppmFigure: τcp = 1 msRODEO-CP removes distortions −→ line-shape fitting −→ δiiBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 19 / 55
  63. 63. Spin diffusion contributionStatic RODEO-CP, τcp = 50 µs.150 100 50 0 ppmFigure: τm = 1s!!#!!Spin diffusion in ferrocene is not sufficient to refill the MAH.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 20 / 55
  64. 64. Spin diffusion contributionStatic RODEO-CP, τcp = 50 µs.150 100 50 0 ppmFigure: τm=5 s!!#!!Spin diffusion in ferrocene is not sufficient to refill the MAH.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 20 / 55
  65. 65. Spin diffusion contributionStatic RODEO-CP, τcp = 50 µs.150 100 50 0 ppmFigure: τm=10 s!!#!!Spin diffusion in ferrocene is not sufficient to refill the MAH.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 20 / 55
  66. 66. Magic Angle Turning contributionCP, MAT@50HzIn black, CP turning at the magic angle (50Hz)static CP, τcp =10 ms−20180 160 140 120 100 80 60 40 20 0 ppmFigure: τcp =50 µs!!#!!!Slow MAT CP is not sufficient to refill the MAH for tCP 1 msBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 21 / 55
  67. 67. Magic Angle Turning contributionCP, MAT@50HzIn black, CP turning at the magic angle (50Hz)static CP, τcp =10 ms−20180 160 140 120 100 80 60 40 20 0 ppmFigure: τcp =150 µs,!!#!!!Slow MAT CP is not sufficient to refill the MAH for tCP 1 msBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 21 / 55
  68. 68. Magic Angle Turning contributionCP, MAT@50HzIn black, CP turning at the magic angle (50Hz)static CP, τcp =10 ms−20180 160 140 120 100 80 60 40 20 0 ppmFigure: τcp =350 µs!!#!!!Slow MAT CP is not sufficient to refill the MAH for tCP 1 msBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 21 / 55
  69. 69. Magic Angle Turning contributionCP, MAT@50HzIn black, CP turning at the magic angle (50Hz)static CP, τcp =10 ms−20180 160 140 120 100 80 60 40 20 0 ppmFigure: τcp =1 ms!!#!!!Slow MAT CP is not sufficient to refill the MAH for tCP 1 msBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 21 / 55
  70. 70. ∼400Hz - MAT RODEO-CPSpinning faster: MAT @414 Hz100 80 60 40 20 ppmCP, τcp =150 µs, MAT @ 414 HzRODEO (MAS@400Hz) improve the line-shape fitting −→ betterresolution in structural parametersBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 22 / 55
  71. 71. ∼400Hz - MAT RODEO-CPSpinning faster: MAT @414 Hz100 80 60 40 20 ppmRODEO-CP, τcp =150 µs, τm = 0.5Tr , MAS @ 414 HzRODEO (MAS@400Hz) improve the line-shape fitting −→ betterresolution in structural parametersBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 22 / 55
  72. 72. ∼400Hz - MAT RODEO-CPSpinning faster: MAT @414 Hz100 80 60 40 20 ppm100 80 60 40 20 ppmFit of RODEO-CP, τcp =150 µs, τm = 0.5Tr , MAS @ 414 HzRODEO (MAS@400Hz) improve the line-shape fitting −→ betterresolution in structural parametersBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 22 / 55
  73. 73. ConclusionsRODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by slow turning at the magic angleSimple and robustSuppress MAH and TOHs for contact times longer ≥ 150 µsEven for very short contact times, RODEO spectra line-shape are veryclose to the theoretical line-shape −→ tensor parameters extractedwith good accuracyOverall a loss of 10% in intensity respect to CP due to π/2-pulseimperfectionsTo increase S/N, adiabatic CP and higher MAS (or other angles) canbe used.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 23 / 55
  74. 74. ConclusionsRODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by slow turning at the magic angleSimple and robustSuppress MAH and TOHs for contact times longer ≥ 150 µsEven for very short contact times, RODEO spectra line-shape are veryclose to the theoretical line-shape −→ tensor parameters extractedwith good accuracyOverall a loss of 10% in intensity respect to CP due to π/2-pulseimperfectionsTo increase S/N, adiabatic CP and higher MAS (or other angles) canbe used.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 23 / 55
  75. 75. ConclusionsRODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by slow turning at the magic angleSimple and robustSuppress MAH and TOHs for contact times longer ≥ 150 µsEven for very short contact times, RODEO spectra line-shape are veryclose to the theoretical line-shape −→ tensor parameters extractedwith good accuracyOverall a loss of 10% in intensity respect to CP due to π/2-pulseimperfectionsTo increase S/N, adiabatic CP and higher MAS (or other angles) canbe used.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 23 / 55
  76. 76. ConclusionsRODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by slow turning at the magic angleSimple and robustSuppress MAH and TOHs for contact times longer ≥ 150 µsEven for very short contact times, RODEO spectra line-shape are veryclose to the theoretical line-shape −→ tensor parameters extractedwith good accuracyOverall a loss of 10% in intensity respect to CP due to π/2-pulseimperfectionsTo increase S/N, adiabatic CP and higher MAS (or other angles) canbe used.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 23 / 55
  77. 77. ConclusionsRODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by slow turning at the magic angleSimple and robustSuppress MAH and TOHs for contact times longer ≥ 150 µsEven for very short contact times, RODEO spectra line-shape are veryclose to the theoretical line-shape −→ tensor parameters extractedwith good accuracyOverall a loss of 10% in intensity respect to CP due to π/2-pulseimperfectionsTo increase S/N, adiabatic CP and higher MAS (or other angles) canbe used.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 23 / 55
  78. 78. ConclusionsRODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by slow turning at the magic angleSimple and robustSuppress MAH and TOHs for contact times longer ≥ 150 µsEven for very short contact times, RODEO spectra line-shape are veryclose to the theoretical line-shape −→ tensor parameters extractedwith good accuracyOverall a loss of 10% in intensity respect to CP due to π/2-pulseimperfectionsTo increase S/N, adiabatic CP and higher MAS (or other angles) canbe used.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 23 / 55
  79. 79. RODEO-CP applied to designed peptides in unorientedmodel membranesDesigned PeptidesKL14in planeKKLLKKAKKLLKK-CONH2KALPtransmembraneGKKLALALALALALALALALKKA-CONH2Model MembranePOPC1-palmitoyl-2-oleoyl-phosphatidylcholineBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 24 / 55
  80. 80. RODEO-CP applied to designed peptides in unorientedmodel membranesDesigned PeptidesKL14in planeKKLLKKAKKLLKK-CONH2KALPtransmembraneGKKLALALALALALALALALKKA-CONH2Model MembranePOPC1-palmitoyl-2-oleoyl-phosphatidylcholineBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 24 / 55
  81. 81. RODEO-CP applied to designed peptides in unorientedmodel membranesDesigned PeptidesKL14in planeKKLLKKAKKLLKK-CONH2KALPtransmembraneGKKLALALALALALALALALKKA-CONH2Model MembranePOPC1-palmitoyl-2-oleoyl-phosphatidylcholineBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 24 / 55
  82. 82. RODEO-CP applied to designed peptides in unorientedmodel membranesσ11, σ22, σ33σ, σ⊥Model!!# $%%%(()350 300 250 200 150 100 50 0 ppm 350 300 250 200 150 100 50 0 ppmKL14 KALPσ33 (ppm) 228.2±0.5 221±4σ22 (ppm) 78±4 77.5±0.3σ11 (ppm) 54±1 55.0±0.2Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 25 / 55
  83. 83. RODEO-CP applied to designed peptides in unorientedmodel membranesσ11, σ22, σ33σ, 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 ppmRODEO-APHH-CP, 50 Hz MAT, τcp = 800 µs, P/L=2/100,298 KKL14 KALPσ (ppm) 72±4 205±4σ⊥ (ppm) 143.5±0.5 78.7±0.3Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 25 / 55
  84. 84. RODEO-CP applied to designed peptides in unorientedmodel membranesσ11, σ22, σ33σ, σ⊥Model!!# $%%%(()σ = σ11cos2αsin2β + σ22sin2αsin2β + σ33cos2βC.Sizun and B.Bechinger, J. Am. Chem. Soc. (2002)0Π2Π3 Π22 ΠΑ0Π2Π3 Π22 ΠΒ100150200Σ−→ α = pitch angle and β= helix tilt (approx: σ33 helixaxis, fast rotational diffusion around ˆn )Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 25 / 55
  85. 85. Helix tilt calculationGraphical solutionKL14: intersection of the surface σ⊥ = f (α, β) with the experimentalplane σ⊥ = 143.5 ppm.0 Π4 Π23 Π22 ΠΑ0Π4Π23 Π22 ΠΒ75100125150Σ ppmBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 26 / 55
  86. 86. Helix tilt calculationGraphical solutionKALP: intersection of the surface σ = f (α, β) with the experimentalplane σ = 205 ppm.0Π4Π23 Π22 ΠΑ0Π4 Π23 Π22 ΠΒ100150200ΣBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 26 / 55
  87. 87. ResultsKALPtopologically open curveβ = f (α).α [0, 2π]β [22.7 − 24.5]°KL14topologically closed curveβ = f (α).α [−63.3, +63.3]°β [70.5, 109.5]°Π2ΠΠ 3 Π22 ΠΑ Π2Π2ΒBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 27 / 55
  88. 88. ResultsKALPtopologically open curveβ = f (α).α [0, 2π]β [22.7 − 24.5]°KL14topologically closed curveβ = f (α).α [−63.3, +63.3]°β [70.5, 109.5]°Π2ΠΠ 3 Π22 ΠΑ Π2Π2ΒΠ2ΠΠ 3 Π22 ΠΑ Π2Π2ΒBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 27 / 55
  89. 89. RODEO-CP applied to PLAH4 in E.coli lipid extractPLAH4 in E.coli total lipid extract15N 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 ppmBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 28 / 55
  90. 90. RODEO-CP applied to PLAH4 in E.coli lipid extractPLAH4 in E.coli total lipid extract15N 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 ppmBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 28 / 55
  91. 91. RODEO-CP applied to PLAH4 in E.coli lipid extractPLAH4 in E.coli total lipid extract15N 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 estimatedvalues (in-plane): σ = 58−81ppm, σ⊥ = 142 − 153 ppm.250 200 150 100 50 0 ppm250 200 150 100 50 0 ppmBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 28 / 55
  92. 92. RODEO-CP applied to PLAH4 in E.coli lipid extractPLAH4 in E.coli total lipid extract15N 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 componentsσ ≈15N σbackboneiso250 200 150 100 50 0 ppm250 200 150 100 50 0 ppm250 200 150 100 50 0 ppm250 200 150 100 50 0 ppmBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 28 / 55
  93. 93. RODEO-CP applied to PLAH4 in E.coli lipid extractPLAH4 in E.coli total lipid extract15N 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 additionalpeaks.250 200 150 100 50 0 ppm250 200 150 100 50 0 ppm250 200 150 100 50 0 ppm250 200 150 100 50 0 ppmH lateral chainsK lateral chainslipids (?)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 28 / 55
  94. 94. RODEO-CP applied to PLAH4 in-vivo E.coliPLAH4 in-vivo E.coli≤0.75mg 15N fully labeledPLAH4∼300 mg bacteria pelletTRIS buffer (pH∼7)no nutrients, no O2RODEO-APHH-CP, 53 Hz MAT, τm = (0.5Tr ± 18%), τCP = 800 µs, 4days acquisition, 298 K.viability tests:no difference wor w/o peptide20% bacteriadiedS/N can beimproved250 200 150 100 50 0 ppmBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 29 / 55
  95. 95. Outline1 IntroductionMotivations2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and Transient Oscillation Holes origins3 A strategy to refill the Magic Angle Hole and Transient Oscillation HolesChanging the shape of the contact pulse4 Another strategy: ROtor Directed Exchange of Orientation (RODEO)RODEO - Theory and method developmentRODEO - Applications5 Biophysical studies of the antimicrobial peptide LAH4LAH4-membrane insertion in presence of citrate6 Future perspectiveBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 30 / 55
  96. 96. LAH4Known propertiesKKALLALALHHL AHLALHLALALKKA-NH2aunstructured in solutionhelical in membrane/micellesaBechinger(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∼5protonation of histidinessurface-associatedpH∼7deprotonation of histidinestransmembraneBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 31 / 55
  97. 97. LAH4 in presence of citrate bufferOriented Solid-State NMR15N single labeled LAH4 in oriented DMPC (P/L=1:50)DMPC= 1,2-dimyristoyl-sn-glycero-3-phosphocholineNo buffer, pH ∼5a200 100 0 ppmFigure: σ ≈80 ppm =⇒In-planeorientation.aWith 10 mM citrate buffer, pH 5200 100 0 ppmFigure: σ ≈ 200 ppm =⇒Transmembrane orientation.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 32 / 55
  98. 98. LAH4 in presence of citrate buffer-IOriented Circular Dichroismabsence of a negative band around 208 nm is an indication of a TMhelixBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 33 / 55
  99. 99. LAH4 in presence of citrate buffer-IOriented Circular Dichroismabsence of a negative band around 208 nm is an indication of a TMhelixBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 33 / 55
  100. 100. Small Angle X-ray Scattering (SAXS)Membrane hydrophobic thicknessEffect of LAH4 on thehydrophobic thickness ofPOPC, POPG andPOPC/POPG vesicles incitrate buffer pH=5Bilayer thickness:dB = 2(zH + 2σH)Membrane hydrophobicthickness:dCC = dB − 10˚A͗f͑q͒͘ϭF͑q͒ϭ2F ͑q͒ϩF ͑q͒, ͑7͒intensity 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, uncorrelatedbilayersI͑q͒ϰ1q2 „͉F͑q͉͒2S͑q͒ϩNdiff͉F͑q͉͒2…. ͑13͒In further context of this paper we will refer to the abovedescribed model as MCG, since it is a combination of MCTand a Gaussian electron density representation of the head-group ͓30͔.A further benefit of this method is that one can derivestructural parameters from simple geometric relationships,without the need of volumetric data as, e.g., in the approachof McIntosh and Simon ͓32͔, or Nagle et al. ͓14͔. For deter-FIG. 1. Electron density profile model ␳(z) as a function ofdistance z from the center of the bilayer, given by a summation oftwo Gaussians ͓see Eq. ͑5͔͒.4002 PRE 62PABST, RAPPOLT, AMENITSCH, AND LAGGNERBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 34 / 55
  101. 101. Small Angle X-ray Scattering (SAXS)Membrane hydrophobic thicknessEffect of LAH4 on thehydrophobic thickness ofPOPC, POPG andPOPC/POPG vesicles incitrate buffer pH=5Bilayer thickness:dB = 2(zH + 2σH)Membrane hydrophobicthickness:dCC = dB − 10˚A͗f͑q͒͘ϭF͑q͒ϭ2F ͑q͒ϩF ͑q͒, ͑7͒intensity 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, uncorrelatedbilayersI͑q͒ϰ1q2 „͉F͑q͉͒2S͑q͒ϩNdiff͉F͑q͉͒2…. ͑13͒In further context of this paper we will refer to the abovedescribed model as MCG, since it is a combination of MCTand a Gaussian electron density representation of the head-group ͓30͔.A further benefit of this method is that one can derivestructural parameters from simple geometric relationships,without the need of volumetric data as, e.g., in the approachof McIntosh and Simon ͓32͔, or Nagle et al. ͓14͔. For deter-FIG. 1. Electron density profile model ␳(z) as a function ofdistance z from the center of the bilayer, given by a summation oftwo Gaussians ͓see Eq. ͑5͔͒.4002 PRE 62PABST, RAPPOLT, AMENITSCH, AND LAGGNERBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 34 / 55
  102. 102. ConclusionsConclusionLAH4 in citrate inserts in a transmembrane manner in DMPC, evenat acidic pH, when histidines are charged.LAH4 assume assumes an in-plane alignment in DMPC when nobuffer is added, in agreement with previous results in other lipids(POPC).The membrane thickening POPC at pH 5 in the presence of citratebuffer, suggest that the peptide inserts in a transmembrane manner.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 35 / 55
  103. 103. ConclusionsConclusionLAH4 in citrate inserts in a transmembrane manner in DMPC, evenat acidic pH, when histidines are charged.LAH4 assume assumes an in-plane alignment in DMPC when nobuffer is added, in agreement with previous results in other lipids(POPC).The membrane thickening POPC at pH 5 in the presence of citratebuffer, suggest that the peptide inserts in a transmembrane manner.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 35 / 55
  104. 104. ConclusionsConclusionLAH4 in citrate inserts in a transmembrane manner in DMPC, evenat acidic pH, when histidines are charged.LAH4 assume assumes an in-plane alignment in DMPC when nobuffer is added, in agreement with previous results in other lipids(POPC).The membrane thickening POPC at pH 5 in the presence of citratebuffer, suggest that the peptide inserts in a transmembrane manner.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 35 / 55
  105. 105. Outline1 IntroductionMotivations2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and Transient Oscillation Holes origins3 A strategy to refill the Magic Angle Hole and Transient Oscillation HolesChanging the shape of the contact pulse4 Another strategy: ROtor Directed Exchange of Orientation (RODEO)RODEO - Theory and method developmentRODEO - Applications5 Biophysical studies of the antimicrobial peptide LAH4LAH4-membrane insertion in presence of citrate6 Future perspectiveBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 36 / 55
  106. 106. Future perspectiveRODEO-Applications-LAH4Improve in vivo E.coli RODEO experimentCompare results obtained in E.coli lipidLAH4 and citrate: open questionspeculiar behavior of the citrate anion or is it general?what is the mechanism?does it affect the antimicrobial activity?Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 37 / 55
  107. 107. Future perspectiveRODEO-Applications-LAH4Improve in vivo E.coli RODEO experimentCompare results obtained in E.coli lipidLAH4 and citrate: open questionspeculiar behavior of the citrate anion or is it general?what is the mechanism?does it affect the antimicrobial activity?Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 37 / 55
  108. 108. Future perspectiveRODEO-Applications-LAH4Improve in vivo E.coli RODEO experimentCompare results obtained in E.coli lipidLAH4 and citrate: open questionspeculiar behavior of the citrate anion or is it general?what is the mechanism?does it affect the antimicrobial activity?Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 37 / 55
  109. 109. Future perspectiveRODEO-Applications-LAH4Improve in vivo E.coli RODEO experimentCompare results obtained in E.coli lipidLAH4 and citrate: open questionspeculiar behavior of the citrate anion or is it general?what is the mechanism?does it affect the antimicrobial activity?Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 37 / 55
  110. 110. Future perspectiveRODEO-Applications-LAH4Improve in vivo E.coli RODEO experimentCompare results obtained in E.coli lipidLAH4 and citrate: open questionspeculiar behavior of the citrate anion or is it general?what is the mechanism?does it affect the antimicrobial activity?Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 37 / 55
  111. 111. AcknowledgmentsThanks to:Prof. Dr.B.BechingerProf. Dr.B.WallaceDr. C. MarquesProf. Dr. WillumeitProf. Dr. N. C.NielsenDr. J.RayaDr. J.HirschingerDr. E.GlattardDr. V.VidovicDr. A.MilesProf. Dr. K.LohnerDr. G.PabstLaboratory of NMRand Biophysics ofMembranesBiocontrol NetworkEU FP6 FundingBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 38 / 55
  112. 112. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAramp CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)CP on ferrocene powder - SetBBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  113. 113. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAs45a CP, tCP = 50 µsrectangular CP performed at tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)a“sφ”tangent-amplitude shapes built on the formulaω1I (t) − ω1S (t) = dIS tanφ(τ2− t)(Hediger et al., 1994)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  114. 114. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAs65 CP, tCP = 50 µsrectangular CP performed at tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)CP on ferrocene powder - SetBBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  115. 115. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAs75 CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)CP on ferrocene powder - SetBBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  116. 116. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAs84.3 CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)CP on ferrocene powder - SetBBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  117. 117. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAs88 CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)CP on ferrocene powder - SetBBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  118. 118. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAs89.5 CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)CP on ferrocene powder - SetBBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  119. 119. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetAs89.9 CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)CP on ferrocene powder - SetBBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  120. 120. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetArectangular CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)CP on ferrocene powder - SetBBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  121. 121. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  122. 122. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  123. 123. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  124. 124. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  125. 125. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  126. 126. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  127. 127. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  128. 128. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  129. 129. Shaped-pulse CP experiments - 50 µsCP on ferrocene powder - SetACP on ferrocene powder - SetBramp CP, tCP = 50 µsrectangular CP, tCP = 10 ms.100 50 ppm 0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 39 / 55
  130. 130. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  131. 131. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  132. 132. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  133. 133. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  134. 134. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  135. 135. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  136. 136. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  137. 137. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  138. 138. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  139. 139. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  140. 140. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  141. 141. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  142. 142. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  143. 143. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  144. 144. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  145. 145. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  146. 146. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  147. 147. Shape variations on static CP experiments - 150 µs1H −13 C CP on ferrocene powder performed with tCP = 150 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 40 / 55
  148. 148. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  149. 149. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  150. 150. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  151. 151. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  152. 152. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  153. 153. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  154. 154. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  155. 155. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  156. 156. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  157. 157. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  158. 158. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  159. 159. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  160. 160. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  161. 161. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  162. 162. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  163. 163. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  164. 164. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  165. 165. Shape variations on static CP experiments - 350 µs1H −13 C CP on ferrocene powder performed with tCP = 350 µs applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 41 / 55
  166. 166. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  167. 167. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  168. 168. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  169. 169. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  170. 170. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  171. 171. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  172. 172. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  173. 173. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  174. 174. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  175. 175. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  176. 176. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  177. 177. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  178. 178. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  179. 179. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  180. 180. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  181. 181. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  182. 182. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  183. 183. Shape variations on static CP experiments - 1 ms1H −13 C CP on ferrocene powder performed with tCP = 1 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 42 / 55
  184. 184. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  185. 185. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  186. 186. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  187. 187. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  188. 188. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  189. 189. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  190. 190. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  191. 191. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  192. 192. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetA). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  193. 193. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  194. 194. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  195. 195. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  196. 196. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  197. 197. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  198. 198. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  199. 199. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  200. 200. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  201. 201. Shape variations on static CP experiments - 3 ms1H −13 C CP on ferrocene powder performed with tCP = 3 ms applyingthe shaped-pulse shown below on 13C (SetB). Confront with rectangularCP performed at tCP = 10 ms.100 50 ppm0,0 1,0Contact Time (arbitrary units)02040608010013Ccontactfield(kHz)Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 43 / 55
  202. 202. Introduction to CP - how?Homonuclear spin couple I-IConservative“Flip-Flop”transitionsHeteronuclear spin couple I-STransitions NOT conservativeDouble rotating frame withωRFI = ωRFSHartmann-Hahn condition:γI ωI = γS ωSBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 44 / 55
  203. 203. Introduction to CP - how?Homonuclear spin couple I-IConservative“Flip-Flop”transitionsHeteronuclear spin couple I-STransitions NOT conservativeDouble rotating frame withωRFI = ωRFSHartmann-Hahn condition:γI ωI = γS ωSBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 44 / 55
  204. 204. Introduction to CP - how?Homonuclear spin couple I-IConservative“Flip-Flop”transitionsHeteronuclear spin couple I-STransitions NOT conservativeDouble rotating frame withωRFI = ωRFSHartmann-Hahn condition:γI ωI = γS ωSBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 44 / 55
  205. 205. Introduction to CP - how?Homonuclear spin couple I-IConservative“Flip-Flop”transitionsHeteronuclear spin couple I-STransitions NOT conservativeDouble rotating frame withωRFI = ωRFSHartmann-Hahn condition:γI ωI = γS ωSBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 44 / 55
  206. 206. RODEO-CP: τm optimizationExperimentalFigure: τm = Tr2CalculatedRODEO-CP: µs, MAT@55Hz;CP with tcp = 10 ms.Random-sampling τm results in a RODEO-CP spectra closer to thequasi-equilibrium line-shape.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 45 / 55
  207. 207. CP dynamicsClassical I-S modelThermodynamic approachI(t) follows a double exponential lawferrocene does not follow this law (M¨uller et al., 1974)MBKE I-I*-S modelBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 46 / 55
  208. 208. CP dynamicsClassical I-S modelThermodynamic approachI(t) follows a double exponential lawferrocene does not follow this law (M¨uller et al., 1974)MBKE I-I*-S modelBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 46 / 55
  209. 209. CP dynamicsClassical I-S modelThermodynamic approachI(t) follows a double exponential lawferrocene does not follow this law (M¨uller et al., 1974)MBKE I-I*-S modelBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 46 / 55
  210. 210. CP dynamicsClassical I-S modelMBKE I-I*-S modelNetwork of coupled I nucleiTransient harmonic oscillationsFigures from Kolodziejski et al., Chem.Rev., 2002Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 46 / 55
  211. 211. CP dynamicsClassical I-S modelMBKE I-I*-S modelNetwork of coupled I nucleiTransient harmonic oscillationsFigures from Kolodziejski et al., Chem.Rev., 2002Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 46 / 55
  212. 212. MKBE modelMKBE SolutionMaster 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 − 12 exp(−Rdf t) − 12 exp−Rdf +Rdp2tcos(bt)damped oscillations: freq. depends on b and decay depends on Rdp ,Rdfthe approach to the final equilibrium is regulated by RdfaM¨uller, Kumar, and Baumann, and Ernst.b|ω1I | = |ω1S |, ω0i ≈ ωRFi ,H(t) = H, b = −γI γS 2r3IS(3 cos2θ − 1), T1ρ = 0,|ω1I | + |ω1S | b Rdp, RdfBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 47 / 55
  213. 213. MKBE modelMKBE SolutionMaster 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 − 12 exp(−Rdf t) − 12 exp−Rdf +Rdp2tcos(bt)damped oscillations: freq. depends on b and decay depends on Rdp ,Rdfthe approach to the final equilibrium is regulated by RdfaM¨uller, Kumar, and Baumann, and Ernst.b|ω1I | = |ω1S |, ω0i ≈ ωRFi ,H(t) = H, b = −γI γS 2r3IS(3 cos2θ − 1), T1ρ = 0,|ω1I | + |ω1S | b Rdp, RdfBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 47 / 55
  214. 214. MKBE modelMKBE SolutionMaster 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 − 12 exp(−Rdf t) − 12 exp−Rdf +Rdp2tcos(bt)damped oscillations: freq. depends on b and decay depends on Rdp ,Rdfthe approach to the final equilibrium is regulated by RdfaM¨uller, Kumar, and Baumann, and Ernst.b|ω1I | = |ω1S |, ω0i ≈ ωRFi ,H(t) = H, b = −γI γS 2r3IS(3 cos2θ − 1), T1ρ = 0,|ω1I | + |ω1S | b Rdp, RdfBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 47 / 55
  215. 215. MKBE modelMKBE SolutionMaster 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 − 12 exp(−Rdf t) − 12 exp−Rdf +Rdp2tcos(bt)damped oscillations: freq. depends on b and decay depends on Rdp ,Rdfthe approach to the final equilibrium is regulated by RdfaM¨uller, Kumar, and Baumann, and Ernst.b|ω1I | = |ω1S |, ω0i ≈ ωRFi ,H(t) = H, b = −γI γS 2r3IS(3 cos2θ − 1), T1ρ = 0,|ω1I | + |ω1S | b Rdp, RdfBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 47 / 55
  216. 216. Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caThe projection of the tensor on the axisparallel to B0, σzz, gives a direct indicationof the σ33 orientation Θ:σzz = σ11sin2Θcos2Φ + σ22sin2Θsin2Φ+ σ33cos2Θ∼ 200 ppm ←→ TRANSMEMBRANE ∼ 80 ppm ←→ IN-PLANEDrawbacksOriented samples challenging to obtainProblematic environmental controlLow filling factor of the coilBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 48 / 55
  217. 217. Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caThe projection of the tensor on the axisparallel to B0, σzz, gives a direct indicationof the σ33 orientation Θ:σzz = σ11sin2Θcos2Φ + σ22sin2Θsin2Φ+ σ33cos2Θ∼ 200 ppm ←→ TRANSMEMBRANE ∼ 80 ppm ←→ IN-PLANEDrawbacksOriented samples challenging to obtainProblematic environmental controlLow filling factor of the coilBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 48 / 55
  218. 218. Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caThe projection of the tensor on the axisparallel to B0, σzz, gives a direct indicationof the σ33 orientation Θ:σzz = σ11sin2Θcos2Φ + σ22sin2Θsin2Φ+ σ33cos2Θ∼ 200 ppm ←→ TRANSMEMBRANE ∼ 80 ppm ←→ IN-PLANEDrawbacksOriented samples challenging to obtainProblematic environmental controlLow filling factor of the coilBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 48 / 55
  219. 219. Oriented SS-NMRMechanically Oriented SamplesB0200 ppm caB080 ppm caThe projection of the tensor on the axisparallel to B0, σzz, gives a direct indicationof the σ33 orientation Θ:σzz = σ11sin2Θcos2Φ + σ22sin2Θsin2Φ+ σ33cos2Θ∼ 200 ppm ←→ TRANSMEMBRANE ∼ 80 ppm ←→ IN-PLANEDrawbacksOriented samples challenging to obtainProblematic environmental controlLow filling factor of the coilBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 48 / 55
  220. 220. Helix tilt calculationGraphical solutionσ = σ11cos2αsin2β + σ22sin2αsin2β + σ33cos2βσ⊥ = σ11(1−cos2αsin2β)+σ22(1−sin2αsin2β)+σ33sin2β2KL14: intersection of the surfaces σ,⊥ = f (α, β) with the experimentalvalues, i.e. the planes σ = 72.1 ppm and σ⊥ = 143.5 ppm.0Π4Π2Π3 Π22 Π Α0Π4Π2Π3 Π22 Π Β100150200Σ0 Π4 Π23 Π22 ΠΑ0Π4Π23 Π22 ΠΒ75100125150Σ ppmBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 49 / 55
  221. 221. Helix tilt calculationGraphical solutionσ = σ11cos2αsin2β + σ22sin2αsin2β + σ33cos2βσ⊥ = σ11(1−cos2αsin2β)+σ22(1−sin2αsin2β)+σ33sin2β2KALP: intersection of the surfaces σ,⊥ = f (α, β) with the experimentalvalues, i.e. the planes σ=205 ppm and σ⊥ = 78.7 ppm.0ΠΠ23 Π22 ΠΑ0ΠΠ23 Π22 ΠΒ100150Σ0Π4Π23 Π22 ΠΑ0Π4 Π23 Π22 ΠΒ100150200ΣBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 49 / 55
  222. 222. SAXS data - POPCPOPCFigure: Diffraction patterns of POPC vesicles with increasing amount of LAH4.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 50 / 55
  223. 223. POPCFigure: Diffraction patterns of POPC vesicles with increasing amount of LAH4.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 51 / 55
  224. 224. POPGFigure: Diffraction patterns of POPG vesicles with increasing amount of LAH4.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 52 / 55
  225. 225. POPC/POPG 3:1Figure: Diffraction patterns of POPC vesicles with increasing amount of LAH4.Barbara Perrone (UdS) 13thSeptember 2011 Thesis defense 53 / 55
  226. 226. Electron Density ProfilesElectron Density Profiles - POPCBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 54 / 55
  227. 227. Electron Density ProfilesElectron Density Profiles - POPGBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 54 / 55
  228. 228. Electron Density ProfilesElectron Density Profiles - POPC/POPGBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 54 / 55
  229. 229. DLS and fluorescence quencingBarbara Perrone (UdS) 13thSeptember 2011 Thesis defense 55 / 55

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