Membrane topologies of the PGLa antimicrobial peptide and a transmembrane anchor sequence by Dynamic Nuclear Polarization/solid-state NMR spectroscopy

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T11878 Solid-state spectroscopy and crystallography

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received: 09 December 2015

accepted: 11 January 2016

Published: 15 February 2016

# Membrane topologies of the PGLa antimicrobial peptide and a transmembrane anchor sequence by Dynamic Nuclear Polarization/ solid-state NMR spectroscopy

Evgeniy Sergeevich Salnikov 1,* , Christopher Aisenbrey 1,* , Fabien Aussenac 2 , Olivier Ouari 3 ,
Hiba Sarrouj 1,4 , Christian Reiter 4 , Paul Tordo 3 , Frank Engelke 4 & Burkhard Bechinger 1

Dynamic Nuclear Polarization (DNP) has been introduced to overcome the sensitivity limitations of
nuclear magnetic resonance (NMR) spectroscopy also of supported lipid bilayers. When investigated by
solid-state NMR techniques the approach typically involves doping the samples with biradicals and their
investigation at cryo-temperatures. Here we investigated the effects of temperature and membrane
hydration on the topology of amphipathic and hydrophobic membrane polypeptides. Although
the antimicrobial PGLa peptide in dimyristoyl phospholipids is particularly sensitive to topological
alterations, the DNP conditions represent well its membrane alignment also found in bacterial lipids at
ambient temperature. With a novel membrane-anchored biradical and purpose-built hardware a 17-fold
enhancement in NMR signal intensity is obtained by DNP which is one of the best obtained for a truly
static matrix-free system. Furthermore, a membrane anchor sequence encompassing 19 hydrophobic
amino acid residues was investigated. Although at cryotemperatures the transmembrane domain
adjusts it membrane tilt angle by about 10 degrees, the temperature dependence of two-dimensional
separated field spectra show that freezing the motions can have beneficial effects for the structural
analysis of this sequence.

Solid-state NMR spectroscopy is unique in providing information at atomic resolution of membrane polypeptides in a native bilayer environment. The technique has been used for investigations of their structure, topology,
dynamics and heterogeneous nature in phospholipid bilayers 1–9 . However, NMR suffers from its inherently low
signal intensity and the investigation of membrane-associated polypeptides turns out particularly difficult due to
the dilution of peptide in lipid (typically around 1-2 mole%) and the increased line width due to inhomogeneous
line broadening. Although these limitations are most pronounced when static samples are investigated the large
anisotropy of NMR chemical shift, dipolar and quadrupolar interactions has been successively used to determine
the structure and/or topology of a number of peptides in oriented lipid bilayers 1,2,8,9 . These could be refined by
additional distance restraints for example from magic angle sample spinning (MAS) solid-state NMR spectra 1,2,8,9 .

During the last years considerable progress has been made in dynamic nuclear polarization (DNP) solid-state
NMR and the technique has been shown to overcome many of the sensitivity limitations of solid-state NMR
spectroscopy 10–13 . During DNP/solid-state NMR experiments the radicals dispersed in the sample result in a
high electron polarization which is transferred to the 1 H bath through microwave irradiation. In a next step
cross-polarization to the heteronuclei assures enhancements of the NMR signal typically by about two orders of
magnitude 10,14 . Also in the case of membrane samples 4,11,15,16 or of membrane proteins in cellular environments
considerable improvements have been obtained 17 .

1University of Strasbourg/CNRS, UMR7177, Chemistry Institute, 67070 Strasbourg, France. 2Bruker BioSpin, 34,
rue de l’Industrie, 67166 Wissembourg, France. 3 Aix-Marseille University, Institut de Chimie Radicalaire, UMR 7273,
Faculté des Sciences, 13397 Marseille, Cédex 20, France. 4 Bruker BioSpin, Silberstreifen, 76287 Rheinstetten,
Germany. * These authors contributed equally to this work. Correspondence and requests for materials should be
[addressed to B.B. (email: bechinge@unistra.fr)](mailto:bechinge@unistra.fr)

Figure 1. Shows 15 N solid-state NMR spectra of 2 mole% [ 15 N-Ala14]-PGLa in uniaxially oriented DMPC/
DMPG 3/1 bilayers (membrane normal parallel to Bo) as a function of temperature and hydration. ( A ) 280
K, ( B ) 243 K, ( C ) 100 K, ( D - F ): 310 K. ( A–C, E ) were hydrated at 93% r.h., ( D ) 100% r.h., and ( F ) dehydrated
during long measurement (likely around 75% r.h.). The spectrum in panel ( C ) was measured under 7 Watts
microwave (MW) irradiation. The gray line in panel ( C ) shows the spectrum of peptide-free POPC vesicles
under microwave irradiation. The position of the PC resonance is labeled with an asterisk.

In order to optimize the magnetization transfer from the unpaired electrons to the nuclei biradicals of suitable geometries have been designed and incorporated into the sample preparation 18–23 . Magnetization transfer
through the cross effect 24–26 requires that the measurements are performed at cryo-temperatures to assure sufficiently long life times of the electron polarization 27–30 . Using magic angle oriented sample spinning good DNP
signal enhancements were obtained for solid-state NMR measurements of oriented membranes when at the same
time it was demonstrated that bilayers made of 1-palmitoyl-2-oleoyl- _sn_ -glycero-3-phosphocholine (POPC) retain
an oriented bilayer morphology even at 100K and in the presence of biradicals 31 .

Following this proof-of-concept study 31 a static flat-coil solid-state NMR/DNP probe head has been designed
and constructed 32 . Using this new equipment here we assessed for the first time peptides of biological relevance
under DNP conditions. We explore if membrane domains retain the overall structure and topology also under
low temperature and DNP conditions, and ask the question if significant DNP efficiencies can be obtained from
such systems when reconstituted in supported lipid bilayers. One test sequence that we investigated is the PGLa
antimicrobial peptide which is particular sensitive to environmental conditions 33 . For comparison a transmembrane helical anchor sequence, which was labeled with 15 N at five consecutive residues was also studied 34 .

Antimicrobial peptides of the magainin class have been investigated extensively by biophysical approaches,
including oriented solid-state NMR spectroscopy, with good evidence that many of them selectively kill bacteria by disrupting their plasma membranes 35–38 . Whereas magainin 2 and related peptides adopt very stable
alignments parallel to the membrane surface under all conditions tested so far 39,40, the PGLa helical sequence
(GMASKAGAIA GKIAKVALKA L-amide) has been shown to be highly affected by the detailed composition of
the phospholipid membrane, its peptide-to-lipid ratio, the hydration level or the presence of other peptides 33,41 .
Therefore, this sequence provides a particularly sensitive test case for the potential effects of low temperature
conditions used in a variety of physical experiments such as x-ray diffraction or DNP/solid-state NMR 4,11,15,16 .

Results and Discussion
Figure 1 shows the proton-decoupled 15 N solid-state NMR spectra of 2 mole% [ 15 N-Ala14]-PGLa reconstituted
into uniaxially oriented 1,2-dimyristoyl- _sn_ -3-phosphocholine/1,2-dimyristoyl- _sn_ -3-phospho-(1′ - _rac_ -glycerol)
(DMPC/DMPG) 3/1 mole/mole bilayers. At 310K and 93% relative humidity (r.h.) a broad signal intensity with
a maximum at 125 ppm is observed (Fig. 1E, line width at half height (LWHH) ca. 80 ppm) in agreement with
previous investigations where PGLa has been found to adopt a tilt angle of 53° 33,42,43 . This tilted configuration has

been suggested to be due to the formation of homo-dimers 42 . When the temperature is decreased below the liquid
crystalline – gel phase transition temperature of both lipids (Tc = 297K and 296K for DMPC and DMPG, respectively) a more homogeneous topology is observed characterized by a 15 N chemical shift maximum of 87 ppm
(Fig. 1A,B; LWHH 23 ppm). This chemical shift coincides with the previously published tilt angle of the PGLa
helix of 81° in liquid crystalline DMPC, DMPC/DMPG or POPC bilayers at lipid-to-peptide ratios < 1 mole% 42,44 .
Importantly the good uniform alignment of PGLa persist at lower temperatures (Fig. 1A–C) and even at sub-gel
phase temperatures of 100K and in the presence of the biradical PyPol-C16 (Fig. 1C; LWHH 30 ppm) 18, typical
conditions of the commercial DNP/solid-state NMR set-up 45 .

Notably, the solid-state NMR spectrum of this sample was recorded under the effect of 7W microwave irradiation. The sample contains only 0.7 mg of peptide labeled with 15 N at a single site and did not provide any apparent
signal intensity without microwave irradiation even after 2 days of acquisition. Therefore, the enhancement factor
could not be determined on this sample, however, spectra from related membranes showed 15 N signal enhancements of 17-fold under static conditions 32 . When it is taken into account that spinning of the sample around the
magic angle (MAS) increases the DNP efficiency several fold 25,26,45 this value is among the best enhancement
factors measured so far for matrix-free systems, including oriented membranes 30–32,46 . This favorable property of
PyPol-C16 in the presence of membranes was made possible through the addition of a palmitoyl chain, which
assures a better distribution in the oriented lipid bilayer samples devoid of bulk water 18,22,23 .

Previously 19 F powder pattern line shapes have been observed for PGLa modified with a CF3-carrying
Phe-derivative at four different Ala or Ile positions when reconstituted into DMPC/DMPG 3/1 and investigated
at 253K or 233K 43 . In mechanically oriented lipid bilayers that are exposed to intense radiofrequency irradiation
during solid-state NMR experiments special consideration has to be given to the exact degree of sample hydration over time within the coil volume (the practical aspects are discussed in reference 47 ). Therefore, spectra of
the same samples shown in Fig. 1A–C,E have also been recorded after equilibration at different degrees of hydration. Whereas Fig. 1D shows a defined intensity at 160 ppm at full hydration 33, a condition difficult to maintain
for oriented lipid bilayers under solid-state NMR conditions 47, a broad range of 15 N-H vector (i.e. PGLa helix)
alignments are observed upon sample dehydration both at 310 K (Fig. 1F) and at 273 K (not shown). Notably, at
the same time the phospholipid membrane remains well aligned with an oriented 31 P solid-state NMR resonance
centered at 35 ppm (not shown).

In order to investigate the temperature dependence of a transmembrane sequence upon temperature variation and under DNP conditions the [ 15 N5]-hΦ 19W peptide (peptide with a core of 19 hydrophobic residues
including a tryptophan; cf. Methods section for details) 34 was investigated by two-dimensional separated local
field spectroscopy where the resolution in the dipolar dimension is enhanced by phase- and frequency switched
Lee-Goldberg decoupling of homonuclear 1 H interactions when at the same time cross polarized with the 15 N
nucleus 48,49 . The spectrum obtained at 100 K and under DNP conditions is shown in Fig. 2D where a partial helical wheel that arises from the 5 labeled residues is obvious.

Interestingly at room temperature the same peptide exhibits a much smaller wheel where many intensities
coincide, indicating that fast motions around the helix long axis occur (Fig. 2A). Furthermore, the center of mass
of the spectra obtained at room temperature and at 100 K is shifted by about 20 ppm. This is probably due to one
or several phase transitions of the lipid bilayer concomitant with changes in the hydrophobic thickness of the
membranes and a smaller tilt angle at the cryo-temperature. At intermediate temperatures where the lipid is in
the gel phase broad intensity distributions are obtained that are difficult to analyze (Fig. 2B,C).

Even though PGLa exhibits a number of membrane topologies the peptide retains an oriented alignment even
at sub-gel temperatures and is thereby amenable to solid-state/DNP investigations with good signal enhancements. It should be noted that here we have chosen a bilayer lipid composition where PGLa is particularly sensitive to topological transitions 33,42,43 . Whereas the gel and sub-gel phases exhibit a more homogenous sample
orientation with better resolved lines also at 93% r.h. (Fig. 1A–C) and thereby more suitable conditions for a
structural analysis, the topological equilibrium is shifted to a more membrane-inserted configuration in the liquid
crystalline bilayer (Fig. 1D,E). Interestingly the low-temperature alignment in DMPC/DMPG closely corresponds
to topologies observed under more physiological conditions (membranes made from POPC or from _E. coli_ lipid
extracts at room temperature) 42,44,50 . Furthermore, in this work we have characterized hydration as an additional
parameter that affects the PGLa topological equilibrium in a sensitive manner. The low temperatures may protect
the sample from dehydration when irradiated with strong radio frequency fields and thereby help to maintain a
good peptide alignment.

For the transmembrane sequence a 20 ppm shift in the 15 N chemical shift position indicates a more upright
tilt angle (by about 10 o ) which is probably due an increased hydrophobic thickness of the bilayer under such
conditions. Whereas the peptide undergoes fast motional averaging and exhibits sharp resonances at room temperature the signals are spread over a wider frequency range under DNP conditions which helps to resolve the
NMR intensities from individual sites (Fig. 2A,D). Therefore, these studies show that freezing such motions can
lead to inhomogeneous line broadening but can also have beneficial effects in spreading the signals over a broader
chemical shift/dipolar coupling range.

Notably, investigating oriented membrane systems at cryo-temperatures by DNP/solid-state NMR is only at
its beginnings and there remains a large potential of improvement in changing for example the lipid composition 51,52 . For example in the case of PGLa it has been demonstrated that a stable in-plane alignment such as the
one observed at low temperatures including DNP conditions (Fig. 1A–C), is obtained in bilayers made from
1-palmitoyl-2-oleoyl- or in _E. coli_ phospholipids, which represent much better the natural lipid composition
than the dimyristoyl-phospholipids 33,50 . Recently, bicellar lipid mixtures have been presented that orient in the
magnetic field of the NMR spectrometer even at temperatures of − 15 °C 53, a development which encourages
further developments to make this alternative approach for oriented membrane preparations also accessible to
DNP solid-state NMR technologies. Furthermore, other sequences exhibit a more stable topology and it can be

Figure 2. PISEMA spectra of [ 15 N5]-hΦ19W carrying five consecutive 15 N labels in uniaxially oriented
POPC bilayers. ( A ) ambient temperature (295K), ( B ) 253K and ( C ) 223K. ( D ) nominal 100K (actual sample
temperature due to microwave irradiation ~180K). ( A - C ) 10 mg of peptide were reconstituted at a peptide-tolipid ratio (P/L) of 1/35 mole/mole supported by ultra-thin glass plates 47 . (D) DNP/solid-state NMR spectrum
of 8.5mg [ 15 N5]-hΦ 19W in the presence of 0.3 mg AMUPol 18 at P/L = 1/20 oriented onto films of high-density
polyethylene 31,32 . The simulations show the intensity distributions at static helical tilt angles of 10 o (red circle)
and 22 o (blue circle). Averaging around the helix long axis results in the collapse of the wheel -like pattern into
its respective center of gravity (green dots).

expected that larger proteins have a more densely packed core 54,55 that is less sensitive to changes in the lipid macroscopic phase properties than the peptides investigated here which further extends the range of applications of
oriented DNP/solid-state NMR spectroscopy.

We estimate that in the experiments presented here it takes several minutes before the final cryo-temperature
is obtained. However, methods for rapid freezing have been developed also for membrane-samples, thus that the
conformational and topological states that rapidly exchange at room temperature are captured and can be investigated by solid-state NMR or electron paramagnetic resonance spectroscopies 56,57 . It should therefore be possible
to take advantage of the increased sensitivity of DNP solid-state NMR also during investigations of the dynamic
features of membrane-associated polypeptides.

Methods
The phospholipids POPC, DMPC and DMPG are from Avanti Polar Lipids (Alabaster, AL). All commercial material was used without further purification.

Peptide sequence and label positions. PGLa (GMASKAGAIA GKIAKVALKA L-amide), labeled with 15 N
at the underlined position, and the hydrophobic peptide [ 15 N5]- hΦ 19W (KKKALLALLALAWALALLALLAKKK)
were prepared by solid phase peptide synthesis as described previously 34 . At five subsequent positions leucine
and alanine labeled with 15 N were incorporated into the peptide (underlined in the above sequence). The PyPol
biradical was prepared as described previously 18 . The preparation of PyPol-C16, a derivative of PyPol bearing a
palmitoyl chain, will be described in detail elsewhere.

Membrane samples for DNP. A homogeneous mixture of lipid, peptide, and radical was obtained by
co-dissolving the membrane components in trifluoroethanol. To prepare oriented-phospholipid membranes,
the solution was spread onto ultra-thin cover glasses for conventional oriented solid-state NMR measurements

(8 × 22 mm, thickness 00; Marienfeld, Lauda-Königshofen, Germany), or for DNP on a High-Density
PolyEthylene (HDPE) film (3 × 8 mm, Goodfellow, Cambridge, UK), dried first in air and followed by high
vacuum overnight 31,47 . Thereafter, the samples were equilibrated during a day in an atmosphere of 93% relative
humidity of D2O/H2O (90/10 by volume). The HDPE film with the membrane layers was carefully folded to fit in
the coil and flattened in between two sapphire plates of 3 × 8 mm and 0.5–0.8 mm thickness 32 .

DNP/solid-state NMR. DNP/solid-state NMR measurements were performed using a Bruker BioSpin
wide-bore 9.4 T magnet and an Avance III solid-state NMR spectrometer equipped with a gyrotron producing 263 GHz irradiation, a microwave transmission line, a cooling unit using liquid nitrogen 45, and a purpose
built static solid-state NMR/DNP probe for oriented samples 32 . An adiabatic cross polarization (CP) pulse
sequence 58 was used with a spectral width of 29.8 kHz and acquisition, cross polarization contact and recycle
delay times of 8.6 ms, 0.3 ms and 3 s, respectively. The 1 H π /2 pulse and SPINAL-64 heteronuclear decoupling field
strengths B1 corresponded to a nutation frequency of 50 kHz 59 . To equilibrate the system before acquisition the
sample was exposed to 16 dummy scans. An exponential line-broadening of 100 Hz was applied before Fourier
transformation.

The DNP/solid-state NMR Polarization inversion spin exchange at the magic angle (PISEMA) spectrum was
recorded on about 9 mg [ 15 N5]-hΦ 19W at a nominal temperature of 100K (where the actual sample temperature
depends on the micro wave (MW) irradiation (~180K)). A step-by-step protocol for setting up and analyzing the
experiment are given in references 49,60 . The effective B1 field strength during the SEMA pulse train was 50 kHz.
During the spin exchange period the amplitude of the 1 H B1 field was decreased to 40.9 kHz to maintain the
Hartmann– Hahn match condition with an effective field along the magic angle of 50 kHz. 128 t1 increments
were recorded by accumulating 64 scans each (total duration 7h; with no significant improvement after 64 t1
increments/3.5 hours).

Conventional (no DNP) CP and PISEMA experiments were performed using a Bruker Avance solid-state
NMR (wide-bore 9.4 T magnet) equipped with efree™ static double resonance bioPE™ probe (Bruker
Spectrospin, Karlsruhe, Germany). The temperature was controlled using a BCU-Xtreme unit (Bruker
Spectrospin, Karlsruhe, Germany). Proton-decoupled 15 N solid-state NMR spectra were acquired using an adiabatic CP pulse sequence, a spectral width, acquisition time, CP contact time and recycle delay of 38.5 kHz,
6.7 ms, 0.8 ms and 3 s, respectively. The 1 H π /2 pulse and SPINAL-64 heteronuclear decoupling field strengths
were 35 kHz 59 . Typically 60000 scans were accumulated, and the spectra were zero filled to 4096 points. A 100 Hz
exponential line- broadening was applied before Fourier transformation. Spectra were externally referenced with
15NH4Cl at 40.0 ppm61.
The field strength during the SEMA pulse train was 60 kHz, 94 t1 increments were recorded by accumulating
2048 scans each (total duration 7 days; with no significant improvement after 64 t1 increments/3 days).

References

1. Hong, M. & de Grado, W. F. Structural basis for proton conduction and inhibition by the influenza M2 protein. _Protein Sci_ 21,
1620–1633 (2012).
2. Gustavsson, M. _et al._ Allosteric regulation of SERCA by phosphorylation-mediated conformational shift of phospholamban. _Proc_
_Natl Acad Sci USA_ 110, 17338–17343 (2013).
3. Baker, L. A. & Baldus, M. Characterization of membrane protein function by solid-state NMR spectroscopy. _Curr Opin Struc Biol_
27, 48–55 (2014).
4. Ong, Y. S., Lakatos, A., Becker-Baldus, J., Pos, K. M. & Glaubitz, C. Detecting Substrates Bound to the Secondary Multidrug Efflux
Pump EmrE by DNP-Enhanced Solid-State NMR. _J Am Chem Soc_ 135, 15754–15762 (2013).
5. Eddy, M. T. _et al._ Magic angle spinning nuclear magnetic resonance characterization of voltage-dependent anion channel gating in
two-dimensional lipid crystalline bilayers. _Biochemistry_ 54, 994–1005 (2015).
6. Gattin, Z. _et al._ Solid-state NMR, electrophysiology and molecular dynamics characterization of human VDAC2. _J Biomol NMR_ 61,
311–320 (2015).
7. Linke, D., Shahid, S., Habeck, M., Bardiaux, B. & van Rossum, B. Membrane-protein structure determination by solid-state NMR
spectroscopy of microcrystals: The autotransport domain of Yersinia YadA. _FASEB J_ 27, 590:591 (2013).
8. Das, N. _et al._ Structure of CrgA, a cell division structural and regulatory protein from Mycobacterium tuberculosis, in lipid bilayers.
_Proc Natl Acad Sci USA_ 112, E119–126 (2015).
9. Michalek, M., Salnikov, E., Werten, S. & Bechinger, B. Structure and topology of the huntingtin 1-17 membrane anchor by a
combined solution and solid-state NMR approach. . _Biophys J_ 105, 699–710 (2013).
10. Can, T. V. _et al._ Overhauser effects in insulating solids. _J Chem Phys_ 141, 064202 (2014).
11. Koers, E. J. _et al._ NMR-based structural biology enhanced by dynamic nuclear polarization at high magnetic field. _J Biomol NMR_ 60,
157–168 (2014).
12. Akbey, U. _et al._ Dynamic nuclear polarization of spherical nanoparticles. _Phys Chem Chem Phys_ 15, 20706–20716 (2013).
13. Rossini, A. J. _et al._ DNP Enhanced NMR Spectroscopy for Pharmaceutical Formulations. _J Am Chem Soc_ 136, 2324–2334 (2014).
14. Rossini, A. J. _et al._ Dynamic nuclear polarization surface enhanced NMR spectroscopy. _Acc Chem Res_ 46, 1942–1951 (2013).
15. Su, Y., Andreas, L. & Griffin, R. G. Magic Angle Spinning NMR of Proteins: High-Frequency Dynamic Nuclear Polarization and H
Detection. _Annu Rev Biochem_ 84, 465–497 (2015).
16. Salnikov, E. S. _et al._ Developing DNP/Solid-State NMR Spectroscopy of Oriented Membranes. _Appl Magn Reson_ 43, 91–106 (2012).
17. Yamamoto, K., Caporini, M. A., Im, S. C., Waskell, L. & Ramamoorthy, A. Cellular solid-state NMR investigation of a membrane
protein using dynamic nuclear polarization. _Biochim Biophys Acta_ 1848, 342–349 (2015).
18. Sauvee, C. _et al._ Highly Efficient, Water-Soluble Polarizing Agents for Dynamic Nuclear Polarization at High Frequency. _Angew_
_Chem Int Ed_ 52, 10858–10861 (2013).
19. Mao, J. F. _et al._ Host-Guest Complexes as Water-Soluble High-Performance DNP Polarizing Agents. _J Am Chem Soc_ 135,
19275–19281 (2013).
20. Dane, E. L. _et al._ Rigid Orthogonal Bis-TEMPO Biradicals with Improved Solubility for Dynamic Nuclear Polarization. _J Org Chem_
77, 1789–1797 (2012).
21. Kubicki, D. J. _et al._ Amplifying Dynamic Nuclear Polarization of Frozen Solutions by Incorporating Dielectric Particles. _J Am Chem_
_Soc_ 136, 15711–15718 (2014).
22. Smith, A. N., Caporini, M. A., Fanucci, G. E. & Long, J. R. A Method for Dynamic Nuclear Polarization Enhancement of Membrane
Proteins. _Angew Chem Int Ed_ 54, 1542–1546 (2015).

23. Fernandez-de-Alba, C. _et al._ Matrix-free DNP-enhanced NMR spectroscopy of liposomes using a lipid-anchored biradical. _Chem_
_Eur J_ 21, 4512–4517 (2015).
24. Can, T. V., Ni, Q. Z. & Griffin, R. G. Mechanisms of dynamic nuclear polarization in insulating solids. _J Magn Reson_ 253, 23–35
(2015).
25. Mentink-Vigier, F. _et al._ Fast passage dynamic nuclear polarization on rotating solids. _J Magn Reson_ 224, 13–21 (2012).
26. Thurber, K. R. & Tycko, R. Theory for cross effect dynamic nuclear polarization under magic-angle spinning in solid state nuclear
magnetic resonance: the importance of level crossings. _J Chem Phys_ 137, 084508 (2012).
27. Thurber, K. R., Potapov, A., Yau, W. M. & Tycko, R. Solid state nuclear magnetic resonance with magic-angle spinning and dynamic
nuclear polarization below 25 K. _J Magn Reson_ 226, 100–106 (2013).
28. Ni, Q. Z. _et al._ High frequency dynamic nuclear polarization. _Acc Chem Res_ 46, 1933–1941 (2013).
29. Akbey, U., Linden, A. H. & Oschkinat, H. High-Temperature Dynamic Nuclear Polarization Enhanced Magic-Angle-Spinning
NMR. _Appl Magn Reson_ 43, 81–90 (2012).
30. Jakdetchai, O. _et al._ Dynamic Nuclear Polarization-Enhanced NMR on Aligned Lipid Bilayers at Ambient Temperature. _J Am Chem_
_Soc_ 136, 15533–15536 (2014).
31. Salnikov, E. _et al._ Solid-state NMR spectroscopy of oriented membrane polypeptides at 100 K with signal enhancement by dynamic
nuclear polarization. _J Am Chem Soc_ 132, 5940–5941 (2010).
32. Salnikov, E. _et al._ Solid-state NMR/Dynamic Nuclear Polarization of planar supported lipid bilayers. _J Phys Chem B_ 119,
14574–14583 (2015).
33. Salnikov, E. & Bechinger, B. Lipid-controlled peptide topology and interactions in bilayers: structural insights into the synergistic
enhancement of the antimicrobial activities of PGLa and magainin 2. _Biophys J_ 100, 1473–1480 (2011).
34. Aisenbrey, C. & Bechinger, B. Tilt and rotational pitch angles of membrane-inserted polypeptides from combined 15N and 2H solidstate NMR spectroscopy. _Biochemistry_ 43, 10502–10512 (2004).
35. Lee, D.-K., Bhunia, A., Kotler, S. A. & Ramamoorthy, A. Detergent-type membrane fragmentation by MSI-78, MSI-367, MSI-594,
and MSI-843 antimicrobial peptides and inhibition by cholesterol: a solid-state nuclear magnetic resonance study. _Biochemistry_ 54,
1897–1907 (2015).
36. Bechinger, B. Detergent-like properties of magainin antibiotic peptides: A 31P solid-state NMR study. _Biochim. Biophys. Acta_ 1712,
101–108 (2005).
37. Ramamoorthy, A. Beyond NMR spectra of antimicrobial peptides: dynamical images at atomic resolution and functional insights.
_Solid State Nucl Magn Reson._ 35, 201–207 (2009).
38. Bechinger, B. The SMART model: Soft Membranes Adapt and Respond, also Transiently, to external stimuli. _J Pept Sci_ 21, 346–355
(2015).
39. Bechinger, B. Insights into the mechanisms of action of host defence peptides from biophysical and structural investigations. _J Pept_
_Sci_ 17, 306–314 (2011).
40. Resende, J. M. _et al._ Membrane interactions of Phylloseptin-1, -2, and -3 peptides by oriented solid-state NMR spectroscopy. _Biophys_
_J_ 107, 901–911 (2014).
41. Glaser, R. W. _et al._ Concentration-dependent realignment of the antimicrobial peptide PGLa in lipid membranes observed by solidstate 19F-NMR. _Biophys J_ 88, 3392–3397 (2005).
42. Strandberg, E., Esteban-Martin, S., Salgado, J. & Ulrich, A. S. Orientation and dynamics of peptides in membranes calculated from
2H-NMR data. _Biophys J_ 96, 3223–3232 (2009).
43. Afonin, S., Grage, S. L., Ieronimo, M., Wadhwani, P. & Ulrich, A. S. Temperature-dependent transmembrane insertion of the
amphiphilic peptide PGLa in lipid bilayers observed by solid state 19F NMR spectroscopy. _J Am Chem Soc_ 130, 16512–16514 (2008).
44. Bechinger, B., Zasloff, M. & Opella, S. J. Structure and Dynamics of the Antibiotic Peptide PGLa in Membranes by Multidimensional
Solution and Solid-State NMR Spectroscopy. _Biophys J_ 74, 981–987 (1998).
45. Rosay, M. _et al._ Solid-state dynamic nuclear polarization at 263 GHz: spectrometer design and experimental results. _Phys Chem_
_Chem Phys_ 12, 5850–5860 (2010).
46. Takahashi, H., Hediger, S. & De Paepe, G. Matrix-free dynamic nuclear polarization enables solid-state NMR C-13-C-13 correlation
spectroscopy of proteins at natural isotopic abundance. _Chem Commun_ 49, 9479–9481 (2013).
47. Aisenbrey, C., Bertani, P. & Bechinger, B. In _Antimicrobial Peptides Methods in Molecular Biology_ eds A. Guiliani & A. C. Rinaldi) Ch.
14, 209–233 (Humana Press, Springer, 2010).
48. Ramamoorthy, A., Wei, Y. & Lee, D. PISEMA Solid-State NMR Spectroscopy. _Annual Reports on NMR Spectroscopy_ 52, 1–52 (2004).
49. Aisenbrey, C., Michalek, M., Salnikov, E. S. & Bechinger, B. In _Lipid-Protein Interactions: Methods and Protocols Methods in_
_Molecular Biology_ (ed J. H. Kleinschmidt) Ch. 16, 357–387 (Springer, 2013).
50. Glattard, E., Salnikov, E. S., Aisenbrey, C. & Bechinger, B. Investigations of the synergistic enhancement of antimicrobial activity in
mixtures of magainin 2 and PGLa. _Biophys Chem, in press_ S0301–4622(15)30005–3, doi: 10.1016/j.bpc.2015.06.002 (2015).
51. Lee, D. K., Kwon, B. S. & Ramamoorthy, A. Freezing point depression of water in phospholipid membranes: a solid-state NMR study.
_Langmuir_ 24, 13598–13604 (2008).
52. Salnikov, E. S. _et al._ Alamethicin topology in phospholipid membranes by oriented solid-state NMR and EPR spectroscopies: A
comparison. _J Phys Chem.B_ 113, 3034–3042 (2009).
53. Yamamoto, K. _et al._ Temperature-resistant bicelles for structural studies by solid-state NMR spectroscopy. _Langmuir_ 31, 1496–1504
(2015).
54. Kamihira, M. _et al._ Structural and orientational constraints of bacteriorhodopsin in purple membranes determined by orientedsample solid-state NMR spectroscopy. _J Struct Biol_ 149, 7–16 (2005).
55. Vogt, T. C. B., Schinzel, S. & Bechinger, B. Biosynthesis of biochemically labelled gramicidins and tyrocidins by Bacillus brevis.
_J Biomol NMR_ 26, 1–11 (2003).
56. Lazo, N. D., Hu, W. & Cross, T. A. Low-temperature solid-state 15 N NMR characterization of polypeptide backbone librations.
_J Magn Reson_ 107, 43–50 (1995).
57. Dzikovski, B. G., Borbat, P. P. & Freed, J. H. Spin-labeled gramicidin a: channel formation and dissociation. _Biophys J_ 87, 3504–3517
(2004).
58. Hediger, S., Meier, B. H. & Ernst, R. R. Adiabatic passage Hartmann-Hahn cross polarization in NMR under magic angle sample
spinning. _Chem Phys Lett_ 240, 449–456 (1995).
59. Fung, B. M., Khitrin, A. K. & Ermolaev, K. An improved broadband decoupling sequence for liquid crystals and solids. _J Magn Reson_
142, 97–101 (2000).
60. Salnikov, E., Aisenbrey, C., Raya, J. & Bechinger, B. In _New Developments in NMR: Advances in Biological Solid-State NMR, Proteins_
_and Membrane-Active Peptides_ eds F. Separovic & A. Naito) Ch. 12, 214–234 (Royal Society of Chemistry, 2014).
61. Bertani, P., Raya, J. & Bechinger, B. 15N chemical shift referencing in solid state NMR. _Solid-state NMR spec_ 61–62, 15–18 (2014).

Acknowledgements
We are grateful to Melanie Rosay, Werner Maas and Alain Belguise for continuous support in making available
DNP time at the Bruker instruments and discussions. We also acknowledge the help by Delphine Hatey during
peptide preparation. CNRS and Bruker Biospin, France have conjointly funded the PhD position of H.S.

Furthermore the financial contributions of the Agence Nationale de la Recherche (projects ProLipIn 10-BLAN731, membraneDNP 12-BSV5-0012, MemPepSyn 14-CE34-0001-01 and the LabEx Chemistry of Complex
Systems 10-LABX-0026_CSC), the University of Strasbourg, the CNRS, the Région Alsace and the RTRA
International Center of Frontier Research in Chemistry are gratefully acknowledged.

Author Contributions
E.S.S. and C.A. prepared samples, performed solid-state NMR experiments and prepared the Figures including
spectral simulations. The DNP experiments were perfomed with the help of F.A. H.S. helped to construct the
prototypic DNP/solid-state NMR probe head under the guidance of F.E. and C.R. and performed preliminary
measurements with the help of F.A. O.O. and P.T. provided the biradicals. B.B. designed the experimental strategy
and wrote the manuscript.

Additional Information
Competing financial interests: The authors declare no competing financial interests.

How to cite this article : Salnikov, E. S. _et al._ Membrane topologies of the PGLa antimicrobial peptide and a
transmembrane anchor sequence by Dynamic Nuclear Polarization / solid-state NMR spectroscopy. _Sci. Rep._ 6,
20895; doi: 10.1038/srep20895 (2016).

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