1. Volume 112, Issue 4, Pages 630-642 (February 2017)
Orientation and Location of the Cyclotide Kalata B1 in Lipid Bilayers Revealed by Solid- State NMR 
Stephan L. Grage, Marc-Antoine Sani, Olivier Cheneval, Sónia Troeira Henriques, Constantin Schalck, Ralf Heinzmann, Joshua S. Mylne, Pavel K. Mykhailiuk, Sergii Afonin, Igor V. Komarov, Frances Separovic, David J. Craik, Anne S. Ulrich 
Biophysical Journal 
Volume 112, Issue 4, Pages (February 2017)
DOI: /j.bpj
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2. Figure 1
Orientational constraints of kalata B1 (sequence in (a)) were obtained using CF3-Bpg as 19F-label (b). The label was substituting one of the residues L2, V4, V10, and V25, all of which are located in the hydrophobic patch (hydrophobic residues indicated in yellow, polar in cyan, anionic in red, cationic in blue, glycines in green, and cystines in white). Using solid-state 19F-NMR, the orientations of the trifluoromethyl director axes, indicated by arrows in two orthogonal views (c and d), were determined individually, and then combined to obtain the overall alignment of the peptide in the lipid bilayer. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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3. Figure 2
Oriented solid-state 19F-NMR spectra of kalata B1, labeled with CF3-Bpg at L2, V4, V10, and V25. Kalata B1 was reconstituted into oriented bilayers composed of (a) POPC at a lipid:peptide ratio of 1:600, (b) POPC at a peptide:lipid ratio of 1:160, (c) POPC/POPE at a peptide:lipid ratio of 1:600, and (d) POPC/POPE at a peptide:lipid ratio of 1:160. The CF3-group 19F-19F dipolar coupling gave rise to a triplet with splitting indicative for the label orientation (indicated above the spectra). The splittings obtained for the POPC samples of peptide:lipid ratio of 1:600 (splittings indicated by dashed vertical lines in a–d) changed slightly in the majority of the labels when the peptide:lipid ratio was increased to 1:160 (b) or in the presence of POPE (c and d) (different splittings indicated above the triplets). All measurements were performed at 35°C. The 19F-NMR spectra were simulated (e–h) using a self-written computer software, to assess the different components and mosaic spread contributing to the lineshapes and leading to a deviation from the expected 1:2:1 triplet. Contributions from peptides in oriented bilayers (black), nonoriented bilayers (dark gray) as well as immobile peptides (light gray) were added such that the sum (e–h) matched the respective experimental lineshape (a–d). A minor oriented component in position V4, as well as the second coexisting component in position V25, are indicated by striped filling. (For more details see Table 1; Supporting Material.)
Biophysical Journal  , DOI: ( /j.bpj )
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4. Figure 3
Oriented solid-state 19F-NMR spectra of CF3-labeled kalata B1 embedded in oriented lipid bilayers at two different sample alignments. The label was introduced as CF3-Bpg substituting at the positions indicated above each column. Kalata B1 in POPC at a peptide:lipid ratio of 1:600 was measured with the sample normal (corresponding to the bilayer normal) placed (a) parallel (at 0°) to the magnetic field, and then placed (b) perpendicular (at 90°) to the magnetic field. The corresponding POPC/POPE samples at a peptide:lipid ratio of 1:600 were measured with the sample normal (c) parallel, and (d) perpendicular to the magnetic field. When turning the samples, in the case of rapid rotational diffusion the triplets are expected to shift to the other side of the isotropic frequency position at −73.4 ppm (indicated by a dashed line) and to be scaled by −1/2. The triplets are indicated above each spectrum. The experimental spectra (a–d) were simulated (e–h) with different components contributing to the lineshape as described in Fig. 2, Table 1, and Supporting Material in more detail.
Biophysical Journal  , DOI: ( /j.bpj )
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5. Figure 4
The orientation of kalata B1 was defined using two Euler angles, τ and ρ, which relate the principal axis system of the moments of inertia (labeled with xMOL, yMOL, zMOL) of the molecule with the laboratory frame (xLAB, yLAB, zLAB), which in turn is aligned with its z-axis along the membrane normal (a). zMOL and xMOL correspond to the smallest and largest moments of inertia, respectively, and yLAB is defined to be perpendicular to both z-axes. τ is the tilt angle between the z-axes, whereas ρ defines the angle between the y-axes, both angles measuring from the laboratory frame to the molecule-fixed frame. The experimental CF3 19F-19F dipolar couplings were compared with the predicted values obtained for varied τ and ρ, and the difference was quantified in terms of an rmsd value. Two 3D structures (1NB1.pdb and 1ZNU.pdb) were used for the analysis of the data obtained for a peptide:lipid ratio of 1:600. The rmsd plot of kalata B1 analyzed with 1NB1.pdb in POPC bilayers (b), and POPC/POPE bilayers (d) show a single rmsd minimum, indicating a well-defined orientation. The rmsd plots of kalata B1 obtained with 1ZNU.pdb in POPC (c), and POPC/POPE (e) exhibit the same global minimum as the respective rmsd plots using 1NB1.pdb, plus a second smaller local minimum. (Each step in the gray scale corresponds to a factor of 1.2 in the rmsd; the rmsd values of the global and second local minima are listed in Table 3 and Table S1.) To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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6. Figure 5
Kalata B1 depicted in the orientation determined from the orientational constraints from 19F-NMR. The lower structures show the kalata B1 orientation in relation to the bilayer leaflet. The top panel gives the view from the aqueous and membrane side. Hydrophobic residues are colored in yellow, polar residues in cyan, glycines in green, and cystines in white. The charged residues E7 and R28 are depicted in red and dark blue, respectively. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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7. Figure 6
The orientation of kalata B1 determined from the 19F-NMR constraints was validated using oriented solid-state 15N-NMR. The 15N-NMR spectrum of uniformly 15N-labeled kalata B1 in oriented 14-O-PC bilayers (a) clearly differs from the powder spectrum of kalata B1 (c), indicating sensitivity to orientation. Using the orientation and order parameters obtained from 19F-NMR analysis, 15N-NMR spectra were predicted for the oriented sample using 3D structures 1NB1.pdb (b, solid line) and 1ZNU.pdb (b, dashed line), using 15N CSA parameters determined from the fitted powder spectrum (d).
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8. Figure 7
De-Paked 2H NMR spectra (0° orientation) of nonoriented multilamellar vesicles composed of d31-POPC (a), POPC/d31-POPE (b), and d31-POPE (c) without (dashed line) and with (solid line) kalata B1 at lipid:peptide ratio of 10:1. Order parameter profiles (d–f) were obtained from the de-Paked spectra (a–c). The error of the order parameter <S(n)> was estimated to be ∼0.01. See Fig. S3 for 2H-NMR spectra before de-Paking. Experiments were performed at 30°C.
Biophysical Journal  , DOI: ( /j.bpj )
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9. Figure 8
Solid-state 31P-NMR spectra of nonoriented, multilamellar vesicles composed of (a) POPC, (b) POPC/POPE, and (c) POPE, without (dotted line) and with (solid line) kalata B1 at a peptide:lipid ratio of 1:10. The experiments were performed at 30°C. 31P-NMR relaxation times (d) T1 and (e) T2 were obtained for vesicles of different lipid composition, without (gray bars) and with (black bars) kalata B1 at a peptide:lipid ratio of 1:10. T1 and T2 relaxation times were measured under 10 kHz MAS, and obtained by fitting the relaxation data of each peak with a single exponential. The asterisk indicates the analyzed lipid component.
Biophysical Journal  , DOI: ( /j.bpj )
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