New methodologies of investigation of model peptides-lipids systems and application to the study of...
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New methodologies of Solid-State NMR and biophysical studies of antimicrobial and designed peptides in model and natural membranes Barbara Perrone Laboratoire de Biophysique et RMN des M´ embranes Universit´ e de Strasbourg, Strasbourg, France September 13th, 2011 Thesis defense
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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.
Transcript of New methodologies of investigation of model peptides-lipids systems and application to the study of...
New methodologies of Solid-State NMR and biophysicalstudies of antimicrobial and designed peptides in model
and natural membranes
Barbara Perrone
Laboratoire de Biophysique et RMN des MembranesUniversite de Strasbourg, Strasbourg, France
September 13th, 2011Thesis defense
Outline
1 IntroductionMotivations
2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and Transient Oscillation Holes origins
3 A strategy to refill the Magic Angle Hole and Transient Oscillation HolesChanging the shape of the contact pulse
4 Another strategy: ROtor Directed Exchange of Orientation (RODEO)RODEO - Theory and method developmentRODEO - Applications
5 Biophysical studies of the antimicrobial peptide LAH4LAH4-membrane insertion in presence of citrate
6 Future perspective
Barbara Perrone (UdS) 13th September 2011 Thesis defense 2 / 55
Outline
1 IntroductionMotivations
2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and Transient Oscillation Holes origins
3 A strategy to refill the Magic Angle Hole and Transient Oscillation HolesChanging the shape of the contact pulse
4 Another strategy: ROtor Directed Exchange of Orientation (RODEO)RODEO - Theory and method developmentRODEO - Applications
5 Biophysical studies of the antimicrobial peptide LAH4LAH4-membrane insertion in presence of citrate
6 Future perspective
Barbara Perrone (UdS) 13th September 2011 Thesis defense 3 / 55
Motivations
Antimicrobial Resistancethreat to public health
Antimicrobial Peptides
Solid-state NMR
2011 E.coli outbreak46 deaths, 3000 persons infected,$2,840,000,000
Mechanisms
SS-NMR methodology
Barbara Perrone (UdS) 13th September 2011 Thesis defense 4 / 55
Outline
1 IntroductionMotivations
2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and Transient Oscillation Holes origins
3 A strategy to refill the Magic Angle Hole and Transient Oscillation HolesChanging the shape of the contact pulse
4 Another strategy: ROtor Directed Exchange of Orientation (RODEO)RODEO - Theory and method developmentRODEO - Applications
5 Biophysical studies of the antimicrobial peptide LAH4LAH4-membrane insertion in presence of citrate
6 Future perspective
Barbara Perrone (UdS) 13th September 2011 Thesis defense 5 / 55
Solid-state NMR - Anisotropy
CSA tensor
σPAF =
σ11 0 00 σ22 00 0 σ33
15N-labeled amide in a helical peptide
σ33 ∼ 200 ppm
σ22 ∼ 85 ppm
σ11 ∼ 65 ppm
Barbara Perrone (UdS) 13th September 2011 Thesis defense 6 / 55
Solid-state NMR - Anisotropy
CSA tensor
σPAF =
σ11 0 00 σ22 00 0 σ33
15N-labeled amide in a helical peptide
σ33 ∼ 200 ppm
σ22 ∼ 85 ppm
σ11 ∼ 65 ppm
Barbara Perrone (UdS) 13th September 2011 Thesis defense 6 / 55
Solid-state NMR - Anisotropy
CSA tensor
σPAF =
σ11 0 00 σ22 00 0 σ33
15N-labeled amide in a helical peptide
σ33 ∼ 200 ppm
σ22 ∼ 85 ppm
σ11 ∼ 65 ppm
Barbara Perrone (UdS) 13th September 2011 Thesis defense 6 / 55
Solid-state NMR - Anisotropy
CSA tensor
300 200 100 0 ppm
!11
!22
!33
σPAF =
σ11 0 00 σ22 00 0 σ33
15N-labeled amide in a helical peptide
σ33 ∼ 200 ppm
σ22 ∼ 85 ppm
σ11 ∼ 65 ppm
Barbara Perrone (UdS) 13th September 2011 Thesis defense 6 / 55
Oriented SS-NMR
Mechanically Oriented Samples
B0
200 ppm ca
B0
80 ppm ca
DrawbacksLow coilfilling-factor due tosupport
Problematicenvironmentalcontrol
Not suitable forcomplex membraneor in cell studies
Barbara Perrone (UdS) 13th September 2011 Thesis defense 7 / 55
Oriented SS-NMR
Mechanically Oriented Samples
B0
200 ppm ca
B0
80 ppm ca
DrawbacksLow coilfilling-factor due tosupport
Problematicenvironmentalcontrol
Not suitable forcomplex membraneor in cell studies
Barbara Perrone (UdS) 13th September 2011 Thesis defense 7 / 55
Oriented SS-NMR
Mechanically Oriented Samples
B0
200 ppm ca
B0
80 ppm ca
DrawbacksLow coilfilling-factor due tosupport
Problematicenvironmentalcontrol
Not suitable forcomplex membraneor in cell studies
Barbara Perrone (UdS) 13th September 2011 Thesis defense 7 / 55
Oriented SS-NMR
Mechanically Oriented Samples
B0
200 ppm ca
B0
80 ppm ca
DrawbacksLow coilfilling-factor due tosupport
Problematicenvironmentalcontrol
Not suitable forcomplex membraneor in cell studies
Barbara Perrone (UdS) 13th September 2011 Thesis defense 7 / 55
Unoriented SS-NMR
Fast uniaxial rotational diffusion around the bilayer normal
Figure: Prongidi-Fix et al., J. Am. Chem. Soc., 2007
15N−KALP inunoriented POPC,310 K
300 200 100 0 ppm
MAH
Distortion at the isotropicfrequency =“Magic AngleHole” (MAH)Major problems withline-shape fitting
Barbara Perrone (UdS) 13th September 2011 Thesis defense 8 / 55
Origins of MAH
Cross-Polarization (CP)
Magnetization transfer: 1H −→13 C ,15N
Dipolar coupling constant:b = −γIγS�
2r3 (3 cos2 θ − 1) b(θ∗) = 0 θ∗ = 54.7° Magic Angle
Chemical Shift Anisotropya: ∆σ ∝ (3 cos2 θ − 1) σ(θ∗) = σiso
ahypothesis: symmetric chemical shift tensor σ� parallel to the dipolar vector
Barbara Perrone (UdS) 13th September 2011 Thesis defense 9 / 55
Static CP under fast uniaxial motion
Static CP of ferrocene
150 100 50 ppm
τcp = 50 µs
Magic Angle Hole (MAH)at the isotropic frequency
Transient 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) 13th September 2011 Thesis defense 10 / 55
Static CP under fast uniaxial motion
Static CP of ferrocene
150 100 50 ppm
τcp = 50 µs
Magic Angle Hole (MAH)at the isotropic frequency
Transient 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) 13th September 2011 Thesis defense 10 / 55
Static CP under fast uniaxial motion
Static CP of ferrocene
150 100 50 ppm
τ cp = 150 µs
Magic Angle Hole (MAH)at the isotropic frequency
Transient 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) 13th September 2011 Thesis defense 10 / 55
Static CP under fast uniaxial motion
Static CP of ferrocene
150 100 50 ppm
τcp = 350 µs
Magic Angle Hole (MAH)at the isotropic frequency
Transient 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) 13th September 2011 Thesis defense 10 / 55
Static CP under fast uniaxial motion
Static CP of ferrocene
150 100 50 ppm
τcp = 1 ms
Magic Angle Hole (MAH)at the isotropic frequency
Transient 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) 13th September 2011 Thesis defense 10 / 55
Static CP under fast uniaxial motion
Static CP of ferrocene
150 100 50 ppm
τcp = 3 ms
Magic Angle Hole (MAH)at the isotropic frequency
Transient 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) 13th September 2011 Thesis defense 10 / 55
Static CP under fast uniaxial motion
Static CP of ferrocene
150 100 50 ppm
τcp = 10 ms
Magic Angle Hole (MAH)at the isotropic frequency
Transient 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) 13th September 2011 Thesis defense 10 / 55
Origin of the Transient Oscillation Holes (TOHs)
Classical ”I-S”model MBKE I-I*-S model
ferroceneMuller et al., Phys. Rev. Lett.,1974
Figures adapted fromKolodziejski et al., Chem.Rev., 2002
Barbara Perrone (UdS) 13th September 2011 Thesis defense 11 / 55
Outline
1 IntroductionMotivations
2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and Transient Oscillation Holes origins
3 A strategy to refill the Magic Angle Hole and Transient Oscillation HolesChanging the shape of the contact pulse
4 Another strategy: ROtor Directed Exchange of Orientation (RODEO)RODEO - Theory and method developmentRODEO - Applications
5 Biophysical studies of the antimicrobial peptide LAH4LAH4-membrane insertion in presence of citrate
6 Future perspective
Barbara Perrone (UdS) 13th September 2011 Thesis defense 12 / 55
Changing the shape of the contact pulse
Shaped-pulse CP Conclusions
tCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/N
tCP =1-3 ms: MAH
Figure: rectangular CP, tCP = 3 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55
Changing the shape of the contact pulse
Shaped-pulse CP Conclusions
tCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/N
tCP =1-3 ms: MAH
Figure: rectangular CP, tCP = 3 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55
Changing the shape of the contact pulse
Shaped-pulse CP Conclusions
tCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/N
tCP =1-3 ms: MAH 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
ramp
Figure: rectangular CP, tCP = 3 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55
Changing the shape of the contact pulse
Shaped-pulse CP Conclusions
tCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/N
tCP =1-3 ms: MAH 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13
C c
on
tact
field
(kH
z)
s45
Figure: rectangular CP, tCP = 3 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55
Changing the shape of the contact pulse
Shaped-pulse CP Conclusions
tCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/N
tCP =1-3 ms: MAH 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13
C c
on
tact
field
(kH
z)
s65
Figure: rectangular CP, tCP = 3 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55
Changing the shape of the contact pulse
Shaped-pulse CP Conclusions
tCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/N
tCP =1-3 ms: MAH 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13
C c
on
tact
field
(kH
z)
s75
Figure: rectangular CP, tCP = 3 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55
Changing the shape of the contact pulse
Shaped-pulse CP Conclusions
tCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/N
tCP =1-3 ms: MAH 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13
C c
on
tact
field
(kH
z)
s84.3
Figure: rectangular CP, tCP = 3 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55
Changing the shape of the contact pulse
Shaped-pulse CP Conclusions
tCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/N
tCP =1-3 ms: MAH 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13
C c
on
tact
field
(kH
z)
s88
Figure: rectangular CP, tCP = 3 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55
Changing the shape of the contact pulse
Shaped-pulse CP Conclusions
tCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/N
tCP =1-3 ms: MAH 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13
C c
on
tact
field
(kH
z)
s89.5
Figure: rectangular CP, tCP = 3 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55
Changing the shape of the contact pulse
Shaped-pulse CP Conclusions
tCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/N
tCP =1-3 ms: MAH 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13
C c
on
tact
field
(kH
z)
s89.9
Figure: rectangular CP, tCP = 3 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55
Changing the shape of the contact pulse
Shaped-pulse CP Conclusions
tCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/N
tCP =1-3 ms: MAH 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
rectangular
Figure: rectangular CP, tCP = 3 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55
Changing the shape of the contact pulse
Shaped-pulse CP Conclusions
tCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/N
tCP =1-3 ms: MAH 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
rectangular
100 50 ppm
Figure: s75 CP, tCP = 50 µs
100 50 ppm
Figure: rectangular CP, tCP = 50 µs
Figure: rectangular CP, tCP = 3 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55
Changing the shape of the contact pulse
Shaped-pulse CP Conclusions
tCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/N
tCP =1-3 ms: MAH 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
rectangular
100 50 ppm
Figure: s88 CP, tCP = 150 µs
100 50 ppm
Figure: rectangular CP, tCP = 150 µs
Figure: rectangular CP, tCP = 3 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55
Changing the shape of the contact pulse
Shaped-pulse CP Conclusions
tCP =50 µs: MAHtCP =150-350 µs: MAH TOHs + 30%S/N
tCP =1-3 ms: MAH 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
rectangular
100 50 ppm
Figure: s75 CP, tCP = 3 ms
100 50 ppm
Figure: rectangular CP, tCP = 3 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 13 / 55
Outline
1 IntroductionMotivations
2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and Transient Oscillation Holes origins
3 A strategy to refill the Magic Angle Hole and Transient Oscillation HolesChanging the shape of the contact pulse
4 Another strategy: ROtor Directed Exchange of Orientation (RODEO)RODEO - Theory and method developmentRODEO - Applications
5 Biophysical studies of the antimicrobial peptide LAH4LAH4-membrane insertion in presence of citrate
6 Future perspective
Barbara Perrone (UdS) 13th September 2011 Thesis defense 14 / 55
ROtor Directed Exchange of Orientation (RODEO)
RODEO-CP pulse sequence
Cross-Polarization
RODEO
Hahn’s echo
Acquisition
!"
!"
#
!!"#
"#"
Barbara Perrone (UdS) 13th September 2011 Thesis defense 15 / 55
ROtor Directed Exchange of Orientation (RODEO)
RODEO-CP pulse sequence
Cross-Polarization
RODEO
Hahn’s echo
Acquisition
!"
!"
#
!!"#
"#"
Barbara Perrone (UdS) 13th September 2011 Thesis defense 15 / 55
ROtor Directed Exchange of Orientation (RODEO)
RODEO-CP pulse sequence
Cross-Polarization
RODEO
Hahn’s echo
Acquisition
!"
!"
#
!!"#
"#"
Barbara Perrone (UdS) 13th September 2011 Thesis defense 15 / 55
ROtor Directed Exchange of Orientation (RODEO)
RODEO-CP pulse sequence
Cross-Polarization
RODEO
Hahn’s echo
Acquisition
!"
!"
#
!!"#
"#"
Barbara Perrone (UdS) 13th September 2011 Thesis defense 15 / 55
ROtor Directed Exchange of Orientation (RODEO)
RODEO-CP pulse sequence
Cross-Polarization
RODEO
Hahn’s echo
Acquisition
!"
!"
#
!!"#
"#"
Barbara Perrone (UdS) 13th September 2011 Thesis defense 15 / 55
MAT(Magic Angle Turning) provide theorientation-exchange
Orientation of the MA cone before and after the mixing time
Figure: before tmix
Barbara Perrone (UdS) 13th September 2011 Thesis defense 16 / 55
MAT(Magic Angle Turning) provide theorientation-exchange
Orientation of the MA cone before and after the mixing time
Figure: after tmix
Barbara Perrone (UdS) 13th September 2011 Thesis defense 16 / 55
MAT(Magic Angle Turning) provide theorientation-exchange
Orientation of the MA cone before and after the mixing time
Figure: intersection (no exchange)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 16 / 55
RODEO-Theory
RODEO Signal:
G (t) =< Sz(tCP) > ·
· exp�
iδω02ωr
�sin2 β2 [sin 2(γ + ωr (t + τm))− sin 2(γ + ωrτm)]
−√2 sin 2β [sin(γ + ωr (t + τm))− sin(γ + ωrτm)]
��
MBKE Solutionab:< Sz(t) >= 1− 1
2 exp(−Rdf t)− 12 exp
�−�Rdf +
Rdp
2
�t�cos(bt)
ϕ = ωrτm between the evolution (CP) and detection (CS) frequencies
aMuller, Kumar, and Baumann, and Ernst (Muller 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) 13th September 2011 Thesis defense 17 / 55
RODEO-CP: effect of τm
RODEO-CP, MAT @ 55 Hz, τcp = 150 µs
As long as τm �= nTr n�N, RODEO refill the MAH and TOH
In black, experimental spectra. In red, theoretical powder-pattern.
−20180 160 140 120 100 80 60 40 20 0 ppm−20180 160 140 120 100 80 60 40 20 0 ppm
Figure: τm = Tr
Barbara Perrone (UdS) 13th September 2011 Thesis defense 18 / 55
RODEO-CP: effect of τm
RODEO-CP, MAT @ 55 Hz, τcp = 150 µs
As long as τm �= nTr n�N, RODEO refill the MAH and TOH
In black, experimental spectra. In red, theoretical powder-pattern.
−20180 160 140 120 100 80 60 40 20 0 ppm−20180 160 140 120 100 80 60 40 20 0 ppm
Figure: τm = 0.1Tr
Barbara Perrone (UdS) 13th September 2011 Thesis defense 18 / 55
RODEO-CP: effect of τm
RODEO-CP, MAT @ 55 Hz, τcp = 150 µs
As long as τm �= nTr n�N, RODEO refill the MAH and TOH
In black, experimental spectra. In red, theoretical powder-pattern.
−20180 160 140 120 100 80 60 40 20 0 ppm−20180 160 140 120 100 80 60 40 20 0 ppm
Figure: τm = 0.2Tr
Barbara Perrone (UdS) 13th September 2011 Thesis defense 18 / 55
RODEO-CP: effect of τm
RODEO-CP, MAT @ 55 Hz, τcp = 150 µs
As long as τm �= nTr n�N, RODEO refill the MAH and TOH
In black, experimental spectra. In red, theoretical powder-pattern.
−20180 160 140 120 100 80 60 40 20 0 ppm−20180 160 140 120 100 80 60 40 20 0 ppm
Figure: τm = 0.3Tr
Barbara Perrone (UdS) 13th September 2011 Thesis defense 18 / 55
RODEO-CP: effect of τm
RODEO-CP, MAT @ 55 Hz, τcp = 150 µs
As long as τm �= nTr n�N, RODEO refill the MAH and TOH
In black, experimental spectra. In red, theoretical powder-pattern.
−20180 160 140 120 100 80 60 40 20 0 ppm−20180 160 140 120 100 80 60 40 20 0 ppm
Figure: τm = 0.4Tr
Barbara Perrone (UdS) 13th September 2011 Thesis defense 18 / 55
RODEO-CP: effect of τm
RODEO-CP, MAT @ 55 Hz, τcp = 150 µs
As long as τm �= nTr n�N, RODEO refill the MAH and TOH
In black, experimental spectra. In red, theoretical powder-pattern.
−20180 160 140 120 100 80 60 40 20 0 ppm−20180 160 140 120 100 80 60 40 20 0 ppm
Figure: τm = 0.5Tr
Barbara Perrone (UdS) 13th September 2011 Thesis defense 18 / 55
RODEO-CP: effect of τm
RODEO-CP, MAT @ 55 Hz, τcp = 150 µs
As long as τm �= nTr n�N, RODEO refill the MAH and TOH
In black, experimental spectra. In red, theoretical powder-pattern.
−20180 160 140 120 100 80 60 40 20 0 ppm−20180 160 140 120 100 80 60 40 20 0 ppm
Figure: τm = 0.6Tr
Barbara Perrone (UdS) 13th September 2011 Thesis defense 18 / 55
RODEO-CP: effect of τm
RODEO-CP, MAT @ 55 Hz, τcp = 150 µs
As long as τm �= nTr n�N, RODEO refill the MAH and TOH
In black, experimental spectra. In red, theoretical powder-pattern.
−20180 160 140 120 100 80 60 40 20 0 ppm−20180 160 140 120 100 80 60 40 20 0 ppm
Figure: τm = 0.7Tr
Barbara Perrone (UdS) 13th September 2011 Thesis defense 18 / 55
RODEO-CP: effect of τm
RODEO-CP, MAT @ 55 Hz, τcp = 150 µs
As long as τm �= nTr n�N, RODEO refill the MAH and TOH
In black, experimental spectra. In red, theoretical powder-pattern.
−20180 160 140 120 100 80 60 40 20 0 ppm−20180 160 140 120 100 80 60 40 20 0 ppm
Figure: τm = 0.8Tr
Barbara Perrone (UdS) 13th September 2011 Thesis defense 18 / 55
RODEO-CP: effect of τm
RODEO-CP, MAT @ 55 Hz, τcp = 150 µs
As long as τm �= nTr n�N, RODEO refill the MAH and TOH
In black, experimental spectra. In red, theoretical powder-pattern.
−20180 160 140 120 100 80 60 40 20 0 ppm−20180 160 140 120 100 80 60 40 20 0 ppm
Figure: τm = 0.9Tr
Barbara Perrone (UdS) 13th September 2011 Thesis defense 18 / 55
RODEO-CP: effect of tCP
RODEO-CP, τmix = Tr/2, MAT @ 50 Hz
In black the experimental spectra, in red the theoretical fit.
150 100 50 0 ppm150 100 50 0 ppm
Figure: τcp = 50µs
RODEO-CP removes distortions −→ line-shape fitting −→ δiiBarbara Perrone (UdS) 13th September 2011 Thesis defense 19 / 55
RODEO-CP: effect of tCP
RODEO-CP, τmix = Tr/2, MAT @ 50 Hz
In black the experimental spectra, in red the theoretical fit.
150 100 50 0 ppm150 100 50 0 ppm
Figure: τcp = 150µs
RODEO-CP removes distortions −→ line-shape fitting −→ δiiBarbara Perrone (UdS) 13th September 2011 Thesis defense 19 / 55
RODEO-CP: effect of tCP
RODEO-CP, τmix = Tr/2, MAT @ 50 Hz
In black the experimental spectra, in red the theoretical fit.
150 100 50 0 ppm150 100 50 0 ppm
Figure: τcp = 350µs
RODEO-CP removes distortions −→ line-shape fitting −→ δiiBarbara Perrone (UdS) 13th September 2011 Thesis defense 19 / 55
RODEO-CP: effect of tCP
RODEO-CP, τmix = Tr/2, MAT @ 50 Hz
In black the experimental spectra, in red the theoretical fit.
150 100 50 0 ppm150 100 50 0 ppm
Figure: τcp = 1 ms
RODEO-CP removes distortions −→ line-shape fitting −→ δiiBarbara Perrone (UdS) 13th September 2011 Thesis defense 19 / 55
Spin diffusion contribution
Static RODEO-CP, τcp = 50 µs.
150 100 50 0 ppm
Figure: τm = 1s
!"
!"
#
!!
Spin diffusion in ferrocene is not sufficient to refill the MAH.
Barbara Perrone (UdS) 13th September 2011 Thesis defense 20 / 55
Spin diffusion contribution
Static RODEO-CP, τcp = 50 µs.
150 100 50 0 ppm
Figure: τm=5 s
!"
!"
#
!!
Spin diffusion in ferrocene is not sufficient to refill the MAH.
Barbara Perrone (UdS) 13th September 2011 Thesis defense 20 / 55
Spin diffusion contribution
Static RODEO-CP, τcp = 50 µs.
150 100 50 0 ppm
Figure: τm=10 s
!"
!"
#
!!
Spin diffusion in ferrocene is not sufficient to refill the MAH.
Barbara Perrone (UdS) 13th September 2011 Thesis defense 20 / 55
Magic Angle Turning contribution
CP, MAT@50Hz
In black, CP turning at the magic angle (50Hz)static CP, τcp =10 ms
−20180 160 140 120 100 80 60 40 20 0 ppm
Figure: τcp =50 µs
!"
!"
#
!!"!
Slow MAT CP is not sufficient to refill the MAH for tCP � 1 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 21 / 55
Magic Angle Turning contribution
CP, MAT@50Hz
In black, CP turning at the magic angle (50Hz)static CP, τcp =10 ms
−20180 160 140 120 100 80 60 40 20 0 ppm
Figure: τcp =150 µs,
!"
!"
#
!!"!
Slow MAT CP is not sufficient to refill the MAH for tCP � 1 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 21 / 55
Magic Angle Turning contribution
CP, MAT@50Hz
In black, CP turning at the magic angle (50Hz)static CP, τcp =10 ms
−20180 160 140 120 100 80 60 40 20 0 ppm
Figure: τcp =350 µs
!"
!"
#
!!"!
Slow MAT CP is not sufficient to refill the MAH for tCP � 1 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 21 / 55
Magic Angle Turning contribution
CP, MAT@50Hz
In black, CP turning at the magic angle (50Hz)static CP, τcp =10 ms
−20180 160 140 120 100 80 60 40 20 0 ppm
Figure: τcp =1 ms
!"
!"
#
!!"!
Slow MAT CP is not sufficient to refill the MAH for tCP � 1 ms
Barbara Perrone (UdS) 13th September 2011 Thesis defense 21 / 55
∼400Hz - MAT RODEO-CP
Spinning faster: MAT @414 Hz
100 80 60 40 20 ppm
CP, τcp =150 µs, MAT @ 414 Hz
RODEO (MAS@400Hz) improve the line-shape fitting −→ betterresolution in structural parameters
Barbara Perrone (UdS) 13th September 2011 Thesis defense 22 / 55
∼400Hz - MAT RODEO-CP
Spinning faster: MAT @414 Hz
100 80 60 40 20 ppm
RODEO-CP, τcp =150 µs, τm = 0.5Tr , MAS @ 414 Hz
RODEO (MAS@400Hz) improve the line-shape fitting −→ betterresolution in structural parameters
Barbara Perrone (UdS) 13th September 2011 Thesis defense 22 / 55
∼400Hz - MAT RODEO-CP
Spinning faster: MAT @414 Hz
100 80 60 40 20 ppm100 80 60 40 20 ppm
Fit of RODEO-CP, τcp =150 µs, τm = 0.5Tr , MAS @ 414 Hz
RODEO (MAS@400Hz) improve the line-shape fitting −→ betterresolution in structural parameters
Barbara Perrone (UdS) 13th September 2011 Thesis defense 22 / 55
Conclusions
RODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by slow turning at the magic angle
Simple and robust
Suppress MAH and TOHs for contact times longer ≥ 150 µs
Even for very short contact times, RODEO spectra line-shape are veryclose to the theoretical line-shape −→ tensor parameters extractedwith good accuracy
Overall a loss of 10% in intensity respect to CP due to π/2-pulseimperfections
To increase S/N, adiabatic CP and higher MAS (or other angles) canbe used.
Barbara Perrone (UdS) 13th September 2011 Thesis defense 23 / 55
Conclusions
RODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by slow turning at the magic angle
Simple and robust
Suppress MAH and TOHs for contact times longer ≥ 150 µs
Even for very short contact times, RODEO spectra line-shape are veryclose to the theoretical line-shape −→ tensor parameters extractedwith good accuracy
Overall a loss of 10% in intensity respect to CP due to π/2-pulseimperfections
To increase S/N, adiabatic CP and higher MAS (or other angles) canbe used.
Barbara Perrone (UdS) 13th September 2011 Thesis defense 23 / 55
Conclusions
RODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by slow turning at the magic angle
Simple and robust
Suppress MAH and TOHs for contact times longer ≥ 150 µs
Even for very short contact times, RODEO spectra line-shape are veryclose to the theoretical line-shape −→ tensor parameters extractedwith good accuracy
Overall a loss of 10% in intensity respect to CP due to π/2-pulseimperfections
To increase S/N, adiabatic CP and higher MAS (or other angles) canbe used.
Barbara Perrone (UdS) 13th September 2011 Thesis defense 23 / 55
Conclusions
RODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by slow turning at the magic angle
Simple and robust
Suppress MAH and TOHs for contact times longer ≥ 150 µs
Even for very short contact times, RODEO spectra line-shape are veryclose to the theoretical line-shape −→ tensor parameters extractedwith good accuracy
Overall a loss of 10% in intensity respect to CP due to π/2-pulseimperfections
To increase S/N, adiabatic CP and higher MAS (or other angles) canbe used.
Barbara Perrone (UdS) 13th September 2011 Thesis defense 23 / 55
Conclusions
RODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by slow turning at the magic angle
Simple and robust
Suppress MAH and TOHs for contact times longer ≥ 150 µs
Even for very short contact times, RODEO spectra line-shape are veryclose to the theoretical line-shape −→ tensor parameters extractedwith good accuracy
Overall a loss of 10% in intensity respect to CP due to π/2-pulseimperfections
To increase S/N, adiabatic CP and higher MAS (or other angles) canbe used.
Barbara Perrone (UdS) 13th September 2011 Thesis defense 23 / 55
Conclusions
RODEORODEO recover the powder pattern line-shape by de-correlating theevolution and detection frequencies by slow turning at the magic angle
Simple and robust
Suppress MAH and TOHs for contact times longer ≥ 150 µs
Even for very short contact times, RODEO spectra line-shape are veryclose to the theoretical line-shape −→ tensor parameters extractedwith good accuracy
Overall a loss of 10% in intensity respect to CP due to π/2-pulseimperfections
To increase S/N, adiabatic CP and higher MAS (or other angles) canbe used.
Barbara Perrone (UdS) 13th September 2011 Thesis defense 23 / 55
RODEO-CP applied to designed peptides in unorientedmodel membranes
Designed Peptides
KL14
in plane
KKLLKKAKKLLKK-CONH2
KALP
transmembrane
GKKLALALALALALALALALKKA-CONH2
Model MembranePOPC1-palmitoyl-2-oleoyl-phosphatidylcholine
Barbara Perrone (UdS) 13th September 2011 Thesis defense 24 / 55
RODEO-CP applied to designed peptides in unorientedmodel membranes
Designed Peptides
KL14
in plane
KKLLKKAKKLLKK-CONH2
KALP
transmembrane
GKKLALALALALALALALALKKA-CONH2
Model MembranePOPC1-palmitoyl-2-oleoyl-phosphatidylcholine
Barbara Perrone (UdS) 13th September 2011 Thesis defense 24 / 55
RODEO-CP applied to designed peptides in unorientedmodel membranes
Designed Peptides
KL14
in plane
KKLLKKAKKLLKK-CONH2
KALP
transmembrane
GKKLALALALALALALALALKKA-CONH2
Model MembranePOPC1-palmitoyl-2-oleoyl-phosphatidylcholine
Barbara Perrone (UdS) 13th September 2011 Thesis defense 24 / 55
RODEO-CP applied to designed peptides in unorientedmodel membranes
σ11, σ22, σ33
σ�, σ⊥
Model!
"
!
"
# $
%&&
%''
%((
)
350 300 250 200 150 100 50 0 ppm 350 300 250 200 150 100 50 0 ppm
KL14 KALPσ33 (ppm) 228.2±0.5 221±4σ22 (ppm) 78±4 77.5±0.3σ11 (ppm) 54±1 55.0±0.2
Barbara Perrone (UdS) 13th September 2011 Thesis defense 25 / 55
RODEO-CP applied to designed peptides in unorientedmodel membranes
σ11, σ22, σ33
σ�, σ⊥
Model!
"
!
"
# $
%&&
%''
%((
)
!50250 200 150 100 50 0 ppm!50250 200 150 100 50 0 ppm !50250 200 150 100 50 0 ppm!50250 200 150 100 50 0 ppm
RODEO-APHH-CP, 50 Hz MAT, τcp = 800 µs, P/L=2/100,298 K
KL14 KALPσ� (ppm) 72±4 205±4σ⊥ (ppm) 143.5±0.5 78.7±0.3
Barbara Perrone (UdS) 13th September 2011 Thesis defense 25 / 55
RODEO-CP applied to designed peptides in unorientedmodel membranes
σ11, σ22, σ33
σ�, σ⊥
Model!
"
!
"
# $
%&&
%''
%((
)
σ� = σ11cos2αsin2β + σ22sin2αsin2β + σ33cos2β
C.Sizun and B.Bechinger, J. Am. Chem. Soc. (2002)
0Π2
Π3 Π2
2 ΠΑ
0
Π2
Π
3 Π2
2 ΠΒ
100
150
200
Σ�
−→ α = pitch angle and β= helix tilt (approx: σ33 � helixaxis, fast rotational diffusion around n )
Barbara Perrone (UdS) 13th September 2011 Thesis defense 25 / 55
Helix tilt calculation
Graphical solution
KL14: intersection of the surface σ⊥ = f (α,β) with the experimentalplane σ⊥ = 143.5 ppm.
0 Π4 Π
2
3 Π2
2 Π
Α
0Π4
Π2
3 Π2
2 Π
Β
75
100
125
150
Σ� �ppm�
Barbara Perrone (UdS) 13th September 2011 Thesis defense 26 / 55
Helix tilt calculation
Graphical solution
KALP: intersection of the surface σ� = f (α,β) with the experimentalplane σ� = 205 ppm.
0Π4Π
2
3 Π2
2 Π
Α
0Π4 Π
2
3 Π2
2 Π
Β
100
150
200
Σ�
Barbara Perrone (UdS) 13th September 2011 Thesis defense 26 / 55
Results
KALPtopologically 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]°
Π2
ΠΠ 3 Π2
2 ΠΑ
� Π2
Π2
Β
Barbara Perrone (UdS) 13th September 2011 Thesis defense 27 / 55
Results
KALPtopologically 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]°
Π2
ΠΠ 3 Π2
2 ΠΑ
� Π2
Π2
Β
Π2
ΠΠ 3 Π2
2 ΠΑ
� Π2
Π2
Β
Barbara Perrone (UdS) 13th September 2011 Thesis defense 27 / 55
RODEO-CP applied to PLAH4 in E.coli lipid extract
PLAH4 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) 13th September 2011 Thesis defense 28 / 55
RODEO-CP applied to PLAH4 in E.coli lipid extract
PLAH4 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) 13th September 2011 Thesis defense 28 / 55
RODEO-CP applied to PLAH4 in E.coli lipid extract
PLAH4 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) 13th September 2011 Thesis defense 28 / 55
RODEO-CP applied to PLAH4 in E.coli lipid extract
PLAH4 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 ≈15
N σbackbone
iso
250 200 150 100 50 0 ppm250 200 150 100 50 0 ppm250 200 150 100 50 0 ppm250 200 150 100 50 0 ppmBarbara Perrone (UdS) 13th September 2011 Thesis defense 28 / 55
RODEO-CP applied to PLAH4 in E.coli lipid extract
PLAH4 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 ppm
H lateral chains
K lateral chains
lipids (?)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 28 / 55
RODEO-CP applied to PLAH4 in-vivo E.coli
PLAH4 in-vivo E.coli
≤0.75mg 15N fully labeledPLAH4
∼300 mg bacteria pellet
TRIS buffer (pH∼7)
no nutrients, no O2
RODEO-APHH-CP, 53 Hz MAT, τm = (0.5Tr ± 18%), τCP = 800 µs, 4days acquisition, 298 K.
viability tests:
no difference wor w/o peptide20% bacteriadied
S/N can beimproved
250 200 150 100 50 0 ppmBarbara Perrone (UdS) 13th September 2011 Thesis defense 29 / 55
Outline
1 IntroductionMotivations
2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and Transient Oscillation Holes origins
3 A strategy to refill the Magic Angle Hole and Transient Oscillation HolesChanging the shape of the contact pulse
4 Another strategy: ROtor Directed Exchange of Orientation (RODEO)RODEO - Theory and method developmentRODEO - Applications
5 Biophysical studies of the antimicrobial peptide LAH4LAH4-membrane insertion in presence of citrate
6 Future perspective
Barbara Perrone (UdS) 13th September 2011 Thesis defense 30 / 55
LAH4
Known properties
KKALLALALHHL AHLALHLALALKKA-NH2a
unstructured in solution
helical in membrane/micelles
aBechinger(1996), Aisenbrey et al. (1996),Vogt et al.(1999),
Kichler et al.(2003), Mason et al. (2006), Kichler et al.(2007),
Prongide-Fix et al. (2007), Marquette et al. (2008)
pH∼5
protonation of histidines
surface-associatedpH∼7
deprotonation of histidines
transmembrane
Barbara Perrone (UdS) 13th September 2011 Thesis defense 31 / 55
LAH4 in presence of citrate buffer
Oriented Solid-State NMR15N single labeled LAH4 in oriented DMPC (P/L=1:50)DMPC= 1,2-dimyristoyl-sn-glycero-3-phosphocholine
No buffer, pH ∼5a
200 100 0 ppm
Figure: σ ≈80 ppm =⇒In-planeorientation.
aDue to the peptide acidity, the pH of
the sample is ∼ 5.
With 10 mM citrate buffer, pH 5
200 100 0 ppm
Figure: σ ≈ 200 ppm =⇒Transmembrane orientation.
Barbara Perrone (UdS) 13th September 2011 Thesis defense 32 / 55
LAH4 in presence of citrate buffer-I
Oriented Circular Dichroism
absence of a negative band around 208 nm is an indication of a TMhelix
Barbara Perrone (UdS) 13th September 2011 Thesis defense 33 / 55
LAH4 in presence of citrate buffer-I
Oriented Circular Dichroism
absence of a negative band around 208 nm is an indication of a TMhelix
Barbara Perrone (UdS) 13th September 2011 Thesis defense 33 / 55
Small Angle X-ray Scattering (SAXS)
Membrane hydrophobic thickness
Effect of LAH4 on thehydrophobic thickness ofPOPC, POPG andPOPC/POPG vesicles incitrate buffer pH=5
Bilayer thickness:dB = 2(zH + 2σH)Membrane hydrophobicthickness:dCC = dB − 10A
! f "q #$!F"q #!2FH"q #"FC"q #, "7#
where the individual components denote the form factor ofthe headgroup
FH"q #!!2%&H'H exp! #&H2 q2
2 " cos"qzH# "8#
and the form factor of the hydrocarbon chains:
FC"q #!!2%&C'C exp! #&C2 q2
2 " . "9#
Equation "7# gives the time averaged form factor of the bi-layer as a continuous function of the scattering vector q.Since the structure factor retained from the Caille theory
considers the lattice disorder, a full q-range description willalso account for the diffuse scattering term in Eq. "3#. Wechoose the discrete formula of the MCT structure factor (9)in the equivalent form of
!s"q #$!S"q #
!N"2*k!1
N#1
"N#k #cos"kqd #
$e#"d/2%#2q2+1,"%k ##"d/2%#2q2+1 , "10#
given in a paper by Lemmich et al. (31). The mean numberof coherent scattering bilayers in the stack is denoted as N,and , is Eulers’ constant. The Caille parameter +1 involvesboth the bending modulus K of lipid bilayers and the bulkmodulus B for compression (7,9),
+h!q2kT
8%!KB, "11#
with
+h!+1h2. "12#
However, during our data analysis we discovered an addi-tional diffuse scattering contribution, which is not describedby the MCT. Its origin is attributed to bilayers with stronglattice defects or unilamellar vesicles, which display neithershort-range nor "quasi-#long-range order. The total scattered
intensity is therefore given by the diffraction of the phospho-lipid multilayers within the quasi-long-range order lattice,plus the additional diffuse scattering of single, uncorrelatedbilayers
I"q #-1q2 „#F"q ##2S"q #"Ndiff#F"q ##2…. "13#
In 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 (30).A further benefit of this method is that one can derive
structural parameters from simple geometric relationships,without the need of volumetric data as, e.g., in the approachof McIntosh and Simon (32), or Nagle et al. (14). For deter-mining the area per lipid, we follow the formalism given byLemmich et al. (33) by calculating the ratio 'r.'H / 'C (seeEq. "5#), which yields
A!1
'CH2" 'r#1 # ! 'rnCe
dC#nHe
dH" , "14#
where nCe is the number of hydrocarbon electrons and nH
e thenumber of headgroup electrons, respectively. The headgroupsize dH can be estimated from the full width at half maxi-mum "FWHM# of the Gaussian, representing the headgroup&(FWHM)H , and the hydrocarbon chain length dC can bederived from
dC!zH#&"FWHM#H
2 "15#
Further parameters of interest are the bilayer thickness
dB!2! zH"&"FWHM#H
2 " ; "16#
the thickness of the water layer,
dW!d#dB , "17#
and the number of interbilayer free water per lipid molecule,
nW*!AdW2VW
"18#
"see, e.g., Refs. (1,14,32)#, where VW is the volume of onewater molecule "approximately 30 Å3#. The total number ofwater molecules including the molecules intercalated into thebilayer, can be estimated from the distance of the headgroupto the bilayer center zH :
nW!A"d/2#zH#
VW. "19#
Finally, the electron density profile can be set on an absolutescale. Here we follow the procedure introduced by Nagle andWiener (34) by calculating the integral
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. "5#).
4002 PRE 62PABST, RAPPOLT, AMENITSCH, AND LAGGNER
Barbara Perrone (UdS) 13th September 2011 Thesis defense 34 / 55
Small Angle X-ray Scattering (SAXS)
Membrane hydrophobic thickness
Effect of LAH4 on thehydrophobic thickness ofPOPC, POPG andPOPC/POPG vesicles incitrate buffer pH=5
Bilayer thickness:dB = 2(zH + 2σH)Membrane hydrophobicthickness:dCC = dB − 10A
! f "q #$!F"q #!2FH"q #"FC"q #, "7#
where the individual components denote the form factor ofthe headgroup
FH"q #!!2%&H'H exp! #&H2 q2
2 " cos"qzH# "8#
and the form factor of the hydrocarbon chains:
FC"q #!!2%&C'C exp! #&C2 q2
2 " . "9#
Equation "7# gives the time averaged form factor of the bi-layer as a continuous function of the scattering vector q.Since the structure factor retained from the Caille theory
considers the lattice disorder, a full q-range description willalso account for the diffuse scattering term in Eq. "3#. Wechoose the discrete formula of the MCT structure factor (9)in the equivalent form of
!s"q #$!S"q #
!N"2*k!1
N#1
"N#k #cos"kqd #
$e#"d/2%#2q2+1,"%k ##"d/2%#2q2+1 , "10#
given in a paper by Lemmich et al. (31). The mean numberof coherent scattering bilayers in the stack is denoted as N,and , is Eulers’ constant. The Caille parameter +1 involvesboth the bending modulus K of lipid bilayers and the bulkmodulus B for compression (7,9),
+h!q2kT
8%!KB, "11#
with
+h!+1h2. "12#
However, during our data analysis we discovered an addi-tional diffuse scattering contribution, which is not describedby the MCT. Its origin is attributed to bilayers with stronglattice defects or unilamellar vesicles, which display neithershort-range nor "quasi-#long-range order. The total scattered
intensity is therefore given by the diffraction of the phospho-lipid multilayers within the quasi-long-range order lattice,plus the additional diffuse scattering of single, uncorrelatedbilayers
I"q #-1q2 „#F"q ##2S"q #"Ndiff#F"q ##2…. "13#
In 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 (30).A further benefit of this method is that one can derive
structural parameters from simple geometric relationships,without the need of volumetric data as, e.g., in the approachof McIntosh and Simon (32), or Nagle et al. (14). For deter-mining the area per lipid, we follow the formalism given byLemmich et al. (33) by calculating the ratio 'r.'H / 'C (seeEq. "5#), which yields
A!1
'CH2" 'r#1 # ! 'rnCe
dC#nHe
dH" , "14#
where nCe is the number of hydrocarbon electrons and nH
e thenumber of headgroup electrons, respectively. The headgroupsize dH can be estimated from the full width at half maxi-mum "FWHM# of the Gaussian, representing the headgroup&(FWHM)H , and the hydrocarbon chain length dC can bederived from
dC!zH#&"FWHM#H
2 "15#
Further parameters of interest are the bilayer thickness
dB!2! zH"&"FWHM#H
2 " ; "16#
the thickness of the water layer,
dW!d#dB , "17#
and the number of interbilayer free water per lipid molecule,
nW*!AdW2VW
"18#
"see, e.g., Refs. (1,14,32)#, where VW is the volume of onewater molecule "approximately 30 Å3#. The total number ofwater molecules including the molecules intercalated into thebilayer, can be estimated from the distance of the headgroupto the bilayer center zH :
nW!A"d/2#zH#
VW. "19#
Finally, the electron density profile can be set on an absolutescale. Here we follow the procedure introduced by Nagle andWiener (34) by calculating the integral
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. "5#).
4002 PRE 62PABST, RAPPOLT, AMENITSCH, AND LAGGNER
Barbara Perrone (UdS) 13th September 2011 Thesis defense 34 / 55
Conclusions
ConclusionLAH4 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) 13th September 2011 Thesis defense 35 / 55
Conclusions
ConclusionLAH4 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) 13th September 2011 Thesis defense 35 / 55
Conclusions
ConclusionLAH4 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) 13th September 2011 Thesis defense 35 / 55
Outline
1 IntroductionMotivations
2 Solid State NMR (SS-NMR)Solid-state NMR and Magic Angle Hole problemMagic Angle Hole and Transient Oscillation Holes origins
3 A strategy to refill the Magic Angle Hole and Transient Oscillation HolesChanging the shape of the contact pulse
4 Another strategy: ROtor Directed Exchange of Orientation (RODEO)RODEO - Theory and method developmentRODEO - Applications
5 Biophysical studies of the antimicrobial peptide LAH4LAH4-membrane insertion in presence of citrate
6 Future perspective
Barbara Perrone (UdS) 13th September 2011 Thesis defense 36 / 55
Future perspective
RODEO-Applications-LAH4
Improve in vivo E.coli RODEO experiment
Compare results obtained in E.coli lipid
LAH4 and citrate: open questions
peculiar behavior of the citrate anion or is it general?
what is the mechanism?
does it affect the antimicrobial activity?
Barbara Perrone (UdS) 13th September 2011 Thesis defense 37 / 55
Future perspective
RODEO-Applications-LAH4
Improve in vivo E.coli RODEO experiment
Compare results obtained in E.coli lipid
LAH4 and citrate: open questions
peculiar behavior of the citrate anion or is it general?
what is the mechanism?
does it affect the antimicrobial activity?
Barbara Perrone (UdS) 13th September 2011 Thesis defense 37 / 55
Future perspective
RODEO-Applications-LAH4
Improve in vivo E.coli RODEO experiment
Compare results obtained in E.coli lipid
LAH4 and citrate: open questions
peculiar behavior of the citrate anion or is it general?
what is the mechanism?
does it affect the antimicrobial activity?
Barbara Perrone (UdS) 13th September 2011 Thesis defense 37 / 55
Future perspective
RODEO-Applications-LAH4
Improve in vivo E.coli RODEO experiment
Compare results obtained in E.coli lipid
LAH4 and citrate: open questions
peculiar behavior of the citrate anion or is it general?
what is the mechanism?
does it affect the antimicrobial activity?
Barbara Perrone (UdS) 13th September 2011 Thesis defense 37 / 55
Future perspective
RODEO-Applications-LAH4
Improve in vivo E.coli RODEO experiment
Compare results obtained in E.coli lipid
LAH4 and citrate: open questions
peculiar behavior of the citrate anion or is it general?
what is the mechanism?
does it affect the antimicrobial activity?
Barbara Perrone (UdS) 13th September 2011 Thesis defense 37 / 55
Acknowledgments
Thanks to:
Prof. Dr.B.Bechinger
Prof. Dr.B.Wallace
Dr. C. Marques
Prof. Dr. Willumeit
Prof. Dr. N. C.Nielsen
Dr. J.Raya
Dr. J.Hirschinger
Dr. E.Glattard
Dr. V.Vidovic
Dr. A.Miles
Prof. Dr. K.Lohner
Dr. G.Pabst
Laboratory of NMRand Biophysics ofMembranes
Biocontrol Network
EU FP6 Funding
Barbara Perrone (UdS) 13th September 2011 Thesis defense 38 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
ramp CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
CP on ferrocene powder - SetB
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
s45a CP, tCP = 50 µs
rectangular CP performed at tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
a“sφ” tangent-amplitude shapes built on the formula
ω1I (t)− ω1S(t) = dIS tan�φ( τ2 − t)
�(Hediger et al., 1994)
CP on ferrocene powder - SetBBarbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
s65 CP, tCP = 50 µs
rectangular CP performed at tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
CP on ferrocene powder - SetB
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
s75 CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
CP on ferrocene powder - SetB
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
s84.3 CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
CP on ferrocene powder - SetB
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
s88 CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
CP on ferrocene powder - SetB
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
s89.5 CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
CP on ferrocene powder - SetB
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
s89.9 CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
CP on ferrocene powder - SetB
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
rectangular CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
CP on ferrocene powder - SetB
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
CP on ferrocene powder - SetB
ramp CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
CP on ferrocene powder - SetB
ramp CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
CP on ferrocene powder - SetB
ramp CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
CP on ferrocene powder - SetB
ramp CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
CP on ferrocene powder - SetB
ramp CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
CP on ferrocene powder - SetB
ramp CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
CP on ferrocene powder - SetB
ramp CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
CP on ferrocene powder - SetB
ramp CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shaped-pulse CP experiments - 50 µs
CP on ferrocene powder - SetA
CP on ferrocene powder - SetB
ramp CP, tCP = 50 µs
rectangular CP, tCP = 10 ms.
100 50 ppm 0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 39 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 150 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 40 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 350 µs
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 41 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 1 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 42 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Shape variations on static CP experiments - 3 ms
1H −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 ppm
0,0 1,0
Contact Time (arbitrary units)
0
20
40
60
80
100
13C
con
tact
field
(kH
z)
Barbara Perrone (UdS) 13th September 2011 Thesis defense 43 / 55
Introduction to CP - how?
Homonuclear spin couple I-I
Conservative“Flip-Flop”transitions
Heteronuclear spin couple I-S
Transitions NOT conservative
Double rotating frame withωRF
I= ωRF
S
Hartmann-Hahn condition:γIωI = γSωS
Barbara Perrone (UdS) 13th September 2011 Thesis defense 44 / 55
Introduction to CP - how?
Homonuclear spin couple I-I
Conservative“Flip-Flop”transitions
Heteronuclear spin couple I-S
Transitions NOT conservative
Double rotating frame withωRF
I= ωRF
S
Hartmann-Hahn condition:γIωI = γSωS
Barbara Perrone (UdS) 13th September 2011 Thesis defense 44 / 55
Introduction to CP - how?
Homonuclear spin couple I-I
Conservative“Flip-Flop”transitions
Heteronuclear spin couple I-S
Transitions NOT conservative
Double rotating frame withωRF
I= ωRF
S
Hartmann-Hahn condition:γIωI = γSωS
Barbara Perrone (UdS) 13th September 2011 Thesis defense 44 / 55
Introduction to CP - how?
Homonuclear spin couple I-I
Conservative“Flip-Flop”transitions
Heteronuclear spin couple I-S
Transitions NOT conservative
Double rotating frame withωRF
I= ωRF
S
Hartmann-Hahn condition:γIωI = γSωS
Barbara Perrone (UdS) 13th September 2011 Thesis defense 44 / 55
RODEO-CP: τm optimization
Experimental
Figure: τm = Tr
2
Calculated
RODEO-CP: µs, MAT@55Hz;CP with tcp = 10 ms.
Random-sampling τm results in a RODEO-CP spectra closer to thequasi-equilibrium line-shape.
Barbara Perrone (UdS) 13th September 2011 Thesis defense 45 / 55
CP dynamics
Classical I-S modelThermodynamic approach
I (t) follows a double exponential law
ferrocene does not follow this law (Muller et al., 1974)
MBKE I-I*-S model
Barbara Perrone (UdS) 13th September 2011 Thesis defense 46 / 55
CP dynamics
Classical I-S modelThermodynamic approach
I (t) follows a double exponential law
ferrocene does not follow this law (Muller et al., 1974)
MBKE I-I*-S model
Barbara Perrone (UdS) 13th September 2011 Thesis defense 46 / 55
CP dynamics
Classical I-S modelThermodynamic approach
I (t) follows a double exponential law
ferrocene does not follow this law (Muller et al., 1974)
MBKE I-I*-S model
Barbara Perrone (UdS) 13th September 2011 Thesis defense 46 / 55
CP dynamics
Classical I-S model
MBKE I-I*-S modelNetwork of coupled I nuclei
Transient harmonic oscillations
Figures from Kolodziejski et al., Chem.Rev., 2002
Barbara Perrone (UdS) 13th September 2011 Thesis defense 46 / 55
CP dynamics
Classical I-S model
MBKE I-I*-S modelNetwork of coupled I nuclei
Transient harmonic oscillations
Figures from Kolodziejski et al., Chem.Rev., 2002
Barbara Perrone (UdS) 13th September 2011 Thesis defense 46 / 55
MKBE model
MKBE 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− 1
2 exp(−Rdf t)− 12 exp
�−�Rdf +
Rdp
2
�t�cos(bt)
damped oscillations: freq. depends on b and decay depends on Rdp ,Rdf
the approach to the final equilibrium is regulated by Rdf
aMuller, Kumar, and Baumann, and Ernst.
b|ω1I | = |ω1S |, ω0i ≈ ωRFi ,H(t) = H, b = − γIγS�2r3
IS
(3 cos2 θ − 1), T1ρ = 0,
|ω1I |+ |ω1S | >> b >> Rdp,Rdf
Barbara Perrone (UdS) 13th September 2011 Thesis defense 47 / 55
MKBE model
MKBE 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− 1
2 exp(−Rdf t)− 12 exp
�−�Rdf +
Rdp
2
�t�cos(bt)
damped oscillations: freq. depends on b and decay depends on Rdp ,Rdf
the approach to the final equilibrium is regulated by Rdf
aMuller, Kumar, and Baumann, and Ernst.
b|ω1I | = |ω1S |, ω0i ≈ ωRFi ,H(t) = H, b = − γIγS�2r3
IS
(3 cos2 θ − 1), T1ρ = 0,
|ω1I |+ |ω1S | >> b >> Rdp,Rdf
Barbara Perrone (UdS) 13th September 2011 Thesis defense 47 / 55
MKBE model
MKBE 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− 1
2 exp(−Rdf t)− 12 exp
�−�Rdf +
Rdp
2
�t�cos(bt)
damped oscillations: freq. depends on b and decay depends on Rdp ,Rdf
the approach to the final equilibrium is regulated by Rdf
aMuller, Kumar, and Baumann, and Ernst.
b|ω1I | = |ω1S |, ω0i ≈ ωRFi ,H(t) = H, b = − γIγS�2r3
IS
(3 cos2 θ − 1), T1ρ = 0,
|ω1I |+ |ω1S | >> b >> Rdp,Rdf
Barbara Perrone (UdS) 13th September 2011 Thesis defense 47 / 55
MKBE model
MKBE 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− 1
2 exp(−Rdf t)− 12 exp
�−�Rdf +
Rdp
2
�t�cos(bt)
damped oscillations: freq. depends on b and decay depends on Rdp ,Rdf
the approach to the final equilibrium is regulated by Rdf
aMuller, Kumar, and Baumann, and Ernst.
b|ω1I | = |ω1S |, ω0i ≈ ωRFi ,H(t) = H, b = − γIγS�2r3
IS
(3 cos2 θ − 1), T1ρ = 0,
|ω1I |+ |ω1S | >> b >> Rdp,Rdf
Barbara Perrone (UdS) 13th September 2011 Thesis defense 47 / 55
Oriented SS-NMR
Mechanically Oriented Samples
B0
200 ppm ca
B0
80 ppm ca
The projection of the tensor on the axisparallel to B0, σzz , gives a direct indicationof the σ33 orientation Θ:
σzz = σ11sin2Θcos2Φ+ σ22sin
2Θsin2Φ
+ σ33cos2Θ
∼ 200 ppm ←→ TRANSMEMBRANE ∼ 80 ppm ←→ IN-PLANE
DrawbacksOriented samples challenging to obtain
Problematic environmental control
Low filling factor of the coil
Barbara Perrone (UdS) 13th September 2011 Thesis defense 48 / 55
Oriented SS-NMR
Mechanically Oriented Samples
B0
200 ppm ca
B0
80 ppm ca
The projection of the tensor on the axisparallel to B0, σzz , gives a direct indicationof the σ33 orientation Θ:
σzz = σ11sin2Θcos2Φ+ σ22sin
2Θsin2Φ
+ σ33cos2Θ
∼ 200 ppm ←→ TRANSMEMBRANE ∼ 80 ppm ←→ IN-PLANE
DrawbacksOriented samples challenging to obtain
Problematic environmental control
Low filling factor of the coil
Barbara Perrone (UdS) 13th September 2011 Thesis defense 48 / 55
Oriented SS-NMR
Mechanically Oriented Samples
B0
200 ppm ca
B0
80 ppm ca
The projection of the tensor on the axisparallel to B0, σzz , gives a direct indicationof the σ33 orientation Θ:
σzz = σ11sin2Θcos2Φ+ σ22sin
2Θsin2Φ
+ σ33cos2Θ
∼ 200 ppm ←→ TRANSMEMBRANE ∼ 80 ppm ←→ IN-PLANE
DrawbacksOriented samples challenging to obtain
Problematic environmental control
Low filling factor of the coil
Barbara Perrone (UdS) 13th September 2011 Thesis defense 48 / 55
Oriented SS-NMR
Mechanically Oriented Samples
B0
200 ppm ca
B0
80 ppm ca
The projection of the tensor on the axisparallel to B0, σzz , gives a direct indicationof the σ33 orientation Θ:
σzz = σ11sin2Θcos2Φ+ σ22sin
2Θsin2Φ
+ σ33cos2Θ
∼ 200 ppm ←→ TRANSMEMBRANE ∼ 80 ppm ←→ IN-PLANE
DrawbacksOriented samples challenging to obtain
Problematic environmental control
Low filling factor of the coil
Barbara Perrone (UdS) 13th September 2011 Thesis defense 48 / 55
Helix tilt calculation
Graphical solution
σ� = σ11cos2αsin2β + σ22sin2αsin2β + σ33cos2β
σ⊥ = σ11(1−cos2αsin2β)+σ22(1−sin
2αsin2β)+σ33sin2β
2
KL14: intersection of the surfaces σ�,⊥ = f (α,β) with the experimentalvalues, i.e. the planes σ� = 72.1 ppm and σ⊥ = 143.5 ppm.
0Π4
Π2
Π
3 Π2
2 Π Α
0Π4Π
2Π
3 Π2
2 Π Β
100
150
200
Σ�
0 Π4 Π
2
3 Π2
2 Π
Α
0Π4
Π2
3 Π2
2 Π
Β
75
100
125
150
Σ� �ppm�
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Helix tilt calculation
Graphical solution
σ� = σ11cos2αsin2β + σ22sin2αsin2β + σ33cos2β
σ⊥ = σ11(1−cos2αsin2β)+σ22(1−sin
2αsin2β)+σ33sin2β
2
KALP: intersection of the surfaces σ�,⊥ = f (α,β) with the experimentalvalues, i.e. the planes σ�=205 ppm and σ⊥ = 78.7 ppm.
0
ΠΠ2
3 Π2
2 ΠΑ
0
ΠΠ2
3 Π2
2 Π
Β
100
150
Σ�
0Π4Π
2
3 Π2
2 Π
Α
0Π4 Π
2
3 Π2
2 Π
Β
100
150
200
Σ�
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SAXS data - POPC
POPC
Figure: Diffraction patterns of POPC vesicles with increasing amount of LAH4.
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POPC
Figure: Diffraction patterns of POPC vesicles with increasing amount of LAH4.
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POPG
Figure: Diffraction patterns of POPG vesicles with increasing amount of LAH4.
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POPC/POPG 3:1
Figure: Diffraction patterns of POPC vesicles with increasing amount of LAH4.
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Electron Density Profiles
Electron Density Profiles - POPC
Figure: POPC density profiles with increasing amount of LAH4.
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Electron Density Profiles
Electron Density Profiles - POPG
Figure: POPC density profiles with increasing amount of LAH4.
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Electron Density Profiles
Electron Density Profiles - POPC/POPG
Figure: POPC/POPG 3:1 density profiles with increasing amount of LAH4.
Barbara Perrone (UdS) 13th September 2011 Thesis defense 54 / 55
DLS and fluorescence quencing
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