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Concentration-Dependent Realignment of the Antimicrobial Peptide PGLa in Lipid Membranes Observed by Solid-State ¹⁹F-NMR

Ralf W. Glaser ^*, Carsten Sachse ^*, Ulrich H. N. Dürr , Parvesh Wadhwani , Sergii Afonin , Erik Strandberg  and Anne S. Ulrich  

^* Institute of Biochemistry and Biophysics, University of Jena, Jena, Germany; Institute of Organic Chemistry, University of Karlsruhe, Karlsruhe, Germany; and Forschungszentrum Karlsruhe, Karlsruhe, Germany

Correspondence: Address reprint requests to Anne S. Ulrich, Institute of Organic Chemistry, University of Karlsruhe, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany. E-mail: anne.ulrich{at}ifia.fzk.de.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS SUPPLEMENTARY MATERIAL ACKNOWLEDGEMENTS REFERENCES

 
The membrane-disruptive antimicrobial peptide PGLa is found to change its orientation in a dimyristoyl-phosphatidylcholine bilayer when its concentration is increased to biologically active levels. The alignment of the -helix was determined by highly sensitive solid-state NMR measurements of ¹⁹F dipolar couplings on CF₃-labeled side chains, and supported by a nonperturbing ¹⁵N label. At a low peptide/lipid ratio of 1:200 the amphiphilic peptide resides on the membrane surface in the so-called S-state, as expected. However, at high peptide concentration (1:50 molar ratio) the helix axis changes its tilt angle from 90° to 120°, with the C-terminus pointing toward the bilayer interior. This tilted "T-state" represents a novel feature of antimicrobial peptides, which is distinct from a membrane-inserted I-state. At intermediate concentration, PGLa is in exchange between the S- and T-state in the timescale of the NMR experiment. In both states the peptide molecules undergo fast rotation around the membrane normal in liquid crystalline bilayers; hence, large peptide aggregates do not form. Very likely the obliquely tilted T-state represents an antiparallel dimer of PGLa that is formed in the membrane at increasing concentration.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS SUPPLEMENTARY MATERIAL ACKNOWLEDGEMENTS REFERENCES

 
PGLa (GMASKAGAIAGKIAKVALKAL-NH₂) is an antibiotic peptide found in frog skin (Hoffmann et al., 1983; Richter et al., 1985; Soravia et al., 1988; Latal et al., 1997). As a member of the magainin family it assumes an amphiphilic -helical structure upon membrane binding (Bechinger et al., 1998). The cationic peptide has a high affinity for bacterial membranes and is able to permeabilize them. A specific receptor is not involved in this antimicrobial action (Wade et al., 1990); hence, it is unlikely that bacteria develop resistance against such peptides. Several different models of antimicrobial action have been proposed (McElhaney and Prenner, 1999; Shai, 1999; Matsuzaki, 1999; Huang, 2000; Zasloff, 2002; Strandberg and Ulrich, 2004). Initially, amphiphilic peptides are supposed to bind flat to the membrane surface in the so-called "S-state". Above a threshold concentration the membrane may then get disrupted nonspecifically by the high surface density, as in the carpet mechanism. Alternatively, a realignment of the peptide molecules into a membrane-inserted "I-state" has been demonstrated, leading to the formation of oligomeric pores. Here, it is appropriate to differentiate between the "barrel-stave" model in which the pore is exclusively made up of peptide molecules, and the "toroidal wormhole" model in which anionic lipids contribute to the pore lining. A better understanding of the structural basis for these different functional mechanisms may help to improve the activity and specificity of antimicrobial peptides.

We have recently developed a highly sensitive solid-state ¹⁹F-NMR strategy for characterizing the orientation and dynamics of membrane-bound peptides, based on collecting a number of orientational constraints (Salgado et al., 2001; Afonin et al., 2003, 2004; Glaser and Ulrich, 2003; Glaser et al., 2004; Ulrich, 2005; Ulrich et al., 2005). The most appropriate label for obtaining these structural data is L-4-trifluoromethyl-phenylglycine (CF₃-Phg), when substituted for a single nonpolar amino acid in the peptide sequence. The anisotropic ¹⁹F-¹⁹F dipolar coupling within the CF₃ group is resolved by ¹⁹F-NMR measurements using macroscopically aligned membrane samples (Glaser et al., 2004). The dipolar splittings from several individual labels serve as experimental constraints to determine the orientation of the peptide within the lipid bilayer. For PGLa reconstituted in DMPC (dimyristoyl-phosphatidylcholine) at a peptide/lipid molar ratio (P/L) of 1:200 we have previously demonstrated that the helix is aligned flat in the plane of the membrane. The tilt angle of 90° between the helix axis and the membrane normal corresponds to the surface-bound S-state, as expected from the amphiphilic character of the peptide. We have now studied PGLa at higher concentration (P/L 1:50), since antimicrobial peptides tend to become biologically active only above such threshold value (Blazyk et al., 2001). Here, we observed an unexpected realignment of the peptide, whereby the helix axis assumes an oblique tilt angle. This new orientation is clearly distinct from a transmembrane alignment, and it does not correspond to an I-state, which would have been expected for such peptides when a pore is formed (Huang, 2000). Instead, it appears that PGLa is able to self-assemble as a dimer on the bilayer surface at high peptide concentration, which would explain how its tilted orientation in the membrane is stabilized as a distinct state.

	MATERIALS AND METHODS

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¹⁹F-NMR analysis
A series of CF₃-Phg labeled PGLa analogs were synthesized, purified, and characterized to be functionally fully intact, as previously reported (Afonin et al., 2003; Glaser et al., 2004). A single label was introduced either at position Ile-9, Ala-10, Ile-13, or Ala-14. Macroscopically oriented samples with a mixture of 0.2–0.5 mg peptide plus lipid in different molar ratios were prepared as previously described, and one-pulse ¹⁹F-NMR spectra were acquired on a 500-MHz wide-bore Unity Inova spectrometer (Varian, Palo Alto, CA) with a ¹⁹F/¹H double-tuned flat-coil probe (Doty Scientific, Columbia, SC) that can be manually rotated and allows ¹H decoupling with 10–15 kHz (Glaser et al., 2004).

¹⁵N-NMR analysis
¹⁵N-labeled PGLa was synthesized by standard Fmoc solid phase synthesis with ¹⁵N-glycine at Gly-11. For the sample with P/L = 1:200, 1 mg of peptide plus 70 mg DMPC were oriented on 25 glass plates, and 2.7 mg PGLa plus 47 mg lipid were used for the P/L 1:50 sample. ¹⁵N-NMR experiments were performed at 50.68 MHz on a Bruker Avance 500 MHz NMR spectrometer with a fixed flat-coil triple-resonance probe (Bruker, Karlsruhe, Germany). We used a ramped cross-polarization sequence with a power of 40 kHz, 2.5 s relaxation delay time, 100 kHz spectral width, 2048 data points, and tppm20 proton decoupling. The contact time was 1 ms in hydrated samples, and 2 ms for the lyophilized peptide. Between 30,000 and 200,000 scans were collected. Spectra were referenced to ¹⁵NH₄NO₃ by setting the signal of solid ¹⁵NH₄Cl to 18.0 ppm. We determined the principal axis values of the ¹⁵N chemical shift anisotropy (CSA) tensor as ₁₁ = 14, ₂₂ = 33, and ₃₃ = 197 ppm by fitting the lineshape to the experimental spectrum of the lyophilized peptide powder. We used an orientation of the ¹⁵N tensor of the labeled glycine with ₃₃ tilted 17° from N-H toward the N-C' bond, and ₁₁ normal to the plane of the peptide bond (Sternberg et al., 2004).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS SUPPLEMENTARY MATERIAL ACKNOWLEDGEMENTS REFERENCES

 
Concentration-dependent realignment of PGLa in DMPC
The one-pulse ¹⁹F-NMR spectra of the four different labeled PGLa analogs were measured at different peptide/lipid ratios in DMPC membranes at 35°C (Fig. 1). At low peptide concentration, i.e., between 1:1000 and 1:200, the dipolar analysis yielded a surface alignment as previously described (Glaser et al., 2004). Upon increasing the P/L to 1:50 and beyond, the chemical shift and dipolar couplings of the labels changed (Table 1). In the following, we will analyze and interpret the structural meaning of these spectral changes, providing new insight into the peptide-lipid interactions of antimicrobial peptides. A similar observation of a concentration-dependent change in NMR parameters had been reported for a 4F-Phg labeled PGLa (Glaser and Ulrich, 2003) as well as a related model peptide K3 (Toke et al., 2004). At that time, no structural interpretation had been possible in these systems.

View larger version (16K): [in this window] [in a new window]   FIGURE 1  Solid-state 19F-NMR spectra of PGLa-Ile13-CF3Phg at 35°C in oriented DMPC membranes with different P/L ratios. The membrane normal is oriented parallel to the B0-field. The dipolar coupling within the CF3-group is read from the splitting of the triplet, and its sign is taken from the chemical shift. The asymmetric shape of the triplet results from differential relaxation properties (Glaser et al., 2004).

Confirmation of realignment in DMPC/DMPG (dimyristoyl-phosphatidylglycerol)
To determine the alignment of PGLa in the membrane from the local orientational constraints of its four labels, the peptide secondary structure has to be verified. Circular dichroism was used to confirm that the helix content of all four analogs is comparable to that of the wild-type peptide in the presence of small unilamellar vesicles at a P/L of 1:50 and 1:200 (see Supplementary Material). Unlike the solid-state ¹⁹F-NMR experiments above, circular dichroism (CD) measurements are performed on vesicles in dilute suspension. Hence, negatively charged lipids are required to attract the cationic PGLa to the membrane. Under those conditions we have previously shown by solution-state ¹⁹F-NMR that the fraction of membrane-bound PGLa increases when negatively charged dimyristoyl-phosphatidylglycerol (DMPG) is mixed into the vesicles of DMPC (Afonin et al., 2003). Once the overall negative charge of the lipids exceeded the positive charge of PGLa (+5 at neutral pH), virtually all PGLa was associated to the membrane and the concentration in bulk solution became immeasurably small.

To test the influence of negative lipid charge on peptide orientation and to justify the CD-derived secondary structure information, we acquired further solid-state ¹⁹F-NMR spectra of a labeled PGLa analog in mixtures of DMPC and DMPG. Measurements in oriented membranes were performed at 1:200 and 1:50 molar peptide/lipid ratio (Fig. 2). The linewidth on these spectra was higher than in samples containing only DMPC. The reason for the line broadening is not clear at this point, but differences in hydration properties and ¹H decoupling are likely. Nevertheless, for 3:1 and 1:1 DMPC/DMPG we found the same qualitative changes in the spectra as in DMPC alone, which is clearly indicative of a reorientation from the S-state at 1:200 to a new state at 1:50. Given that the conformation of PGLa remains -helical in all cases, the observed changes in the ¹⁹F-NMR spectra must therefore be attributed to a concentration-induced realignment of PGLa in the membrane.

View larger version (25K): [in this window] [in a new window]   FIGURE 2  19F-NMR spectra of PGLa (as in Fig. 1) in oriented membranes with different mixtures of DMPC and DMPG.

View larger version (31K): [in this window] [in a new window]   FIGURE 3  Orientation of the PGLa helix in a DMPC bilayer at a 1:200 (upper panels) and 1:50 (lower panels) molar peptide/lipid ratio. The contour plots A–D show the sum of squared deviations between the experimental data and calculated values as a function of the helix tilt angle and the azimuthal rotation , with best-fitting orientations in dark red. (A and B) Summary of the 19F-NMR analysis based on four CF3-Phg labels, and (C and D) a single 15N label. The corresponding 15N spectra are shown in E and F, with the powder lineshape of the lyophilized peptide superimposed.

Peptide mobility in the membrane
The orientational analysis above yielded an order parameter of S_mol 0.6 for PGLa in both the S- and the T-states in liquid crystalline DMPC. On a scale of 1.0 (no averaging) to 0.0 (isotropic tumbling) this value accounts for both the global orientational mobility of the peptide with respect to the membrane normal and the local wobbling motion of the label.

Rotational diffusion of PGLa around the membrane normal is not visible in the spectra acquired with the membrane normal aligned parallel to the static magnetic field direction, but it is revealed when the sample is positioned at a 90° angle, i.e., with the membrane normal perpendicular to the field (Salgado et al., 2001; Ulrich, 2004; Glaser et al., 2004). All spectra at P/L ratios of 1:200 and 1:50 were found to scale around the isotropic chemical shift by a factor of –1/2 compared to the respective spectrum acquired at 0° angle, and no powder pattern or signal broadening was observed (Fig. 4). This indicates that all NMR interactions are averaged around the sample axis on the timescale of the experiment (10 kHz spectral width). Thus, the PGLa molecules rotate freely around the membrane normal, in both the S- and T-states.

View larger version (13K): [in this window] [in a new window]   FIGURE 4  19F-NMR spectra of PGLa (as in Fig. 1) at a P/L of 1:50 in oriented DMPC membranes acquired with two different sample orientations. Upper spectrum with the membrane normal aligned perpendicular to the magnetic field (90° tilt), and lower spectrum with the membrane normal parallel to the magnetic field (0° tilt). In addition, the idealized triplet structure of the spectrum along with the isotropic chemical shift of CF3-Phg (dotted line) are indicated.

Verification of the peptide tilt angle by ¹⁵N-NMR
To decide whether the observed spectral changes at high peptide concentration are genuinely due to an oblique tilt of PGLa, we studied a peptide labeled with ¹⁵N at Gly-11. ¹⁵N-NMR experiments have a much lower sensitivity compared to ¹⁹F-NMR experiments (Afonin et al., 2003), but we can be sure that this label does not perturb the local peptide conformation nor its interactions with other molecules. At low peptide concentration (P/L = 1:200) the ¹⁵N signal of an oriented sample is at 40 ppm, and at high concentration (1:50) it moves to 68 ppm (Fig. 3, E and F). To find out which alignments of the peptide helix are compatible with these data, the principal axis values of the ¹⁵N CSA tensor need to be known. As Gly differs slightly from other amino acids (Sternberg et al., 2004), the values were determined from the lyophilized peptide powder, which is shown superimposed over the oriented spectra.

Orientational analysis of the ¹⁵N data in terms of yielded a tilt angle of 90° for a P/L of 1:200, confirming the S-state, as expected (Fig. 3 C). At a high peptide concentration of 1:50, we determined a helix tilt of about 65° or 115°, which is also in full agreement with the ¹⁹F analysis (Fig. 3 D). This orientational analysis was performed with S_mol = 0.7. For the ¹⁵N label in the backbone, a slightly higher order parameter than 0.6 was chosen to compensate for any extra wobble of the CF₃-Phg side chain and any motion in the lyophilized peptide. The signal lies close to (but not at) the isotropic position of ¹⁵N, but free tumbling or micellarization of the peptide can be excluded in view of the ¹⁹F- and ³¹P-NMR data on equivalent samples. For any reasonable choice of 0.5 < S_mol < 1 only an oblique helix tilt can explain the ¹⁵N data at P/L = 1:50. The evaluation of an oblique helix tilt angle thus unequivocally confirms the self-consistent picture derived above by ¹⁹F-NMR.

We note that the ¹⁵N-NMR signal at 40 ppm of the 1:50 sample is significantly broader than the signal of the 1:200 sample at 68 ppm. This is an inevitable consequence of the differential effect of the mosaic spread on the ¹⁵N chemical shift in the different CSA tensor orientations. Namely, the same mosaic spread produces an apparent linewidth that is 3 times smaller near the high-field and low-field edge of the CSA range (S-state) than in the middle of the CSA range (T-state).

Further evidence for a realignment of PGLa from an S-state to a T-state stems from analyzing the epimeric byproducts of our peptide synthesis, namely those PGLa analogs carrying a D-CF₃-Phg label (see Supplementary Material). The bulky and stiff D-amino acid is expected to cause slight changes in the structure and in the hydrophobic surface of the peptide; therefore, the dipolar couplings only provide qualitative information about the orientation of the peptide (Glaser et al., 2004). Nevertheless, these measurements also confirmed the obliquely tilted orientation of PGLa at high concentration in the membrane, and they clearly excluded the possibility of a transmembrane orientation of the PGLa helix.

Interpretation in terms of peptide dimerization
Notably, the concentration-dependent series of experiments never showed a gradual shift of the signals (e.g., Fig. 1) (as also observed in Glaser and Ulrich, 2003). At peptide/lipid ratios around 1:100, the NMR signal deteriorates because of exchange between the two populations in the intermediate timescale. This is taken as evidence for the presence of two distinct populations of peptides in different orientational states, separated by an energy barrier. A tilted state with = 120° would not seem to be a particularly favorable alignment for a monomer of PGL, which is already perfectly stable in a flat alignment (S-state). Hence, the change from the S- to the T-state must involve more than just a change in orientation with respect to the membrane. Significant conformational changes were excluded by CD experiments, and are very unlikely since our NMR data fits well with a helical conformation in both states. The formation of large peptide aggregates can also be excluded in view of the observed fast rotation around the membrane normal and the same S_mol at low concentration. Therefore, although we have no direct experimental evidence, the most probable explanation for the occurrence of the T-state is the formation of peptide dimers upon increasing the PGLa concentration in the membrane. Indeed, PGLa is known to form dimers in a lipid monolayer at the air-water interface (Konovalov et al., 2002). Dimerization in the membrane has even been described for related antimicrobial peptides: REDOR distance measurements on the model peptide K3 showed parallel dimers (Toke et al., 2004), transferred NOE of a magainin analog indicated antiparallel dimers (Wakamatsu et al., 2002), and PGLa was found to associate with magainin as an antiparallel heterodimer (Hara et al., 2001). The tilt angle of such peptide dimers in the membrane has not yet been determined, as these experiments provided distance constraints but no orientational information. In our case of PGLa, the homodimers in DMPC must be rotationally symmetric with respect to the membrane normal, since we observe only a single set of NMR signals. The tilt of the monomer is then a consequence of the dimer orientation according to its overall amphiphilic shape. Self-assembly of PGLa into antiparallel dimers may thus account for the observation of the distinctly tilted T-state, as schematically illustrated in Fig. 5.

View larger version (51K): [in this window] [in a new window]   FIGURE 5  Illustration of the observed realignment of PGLa in a DMPC membrane. At low peptide concentration, the amphiphilic helix lies flat on the surface in the S-state. With increasing concentration, it assumes a tilted T-state, which appears to involve the formation of antiparallel peptide dimers.

With a small number of orientational constraints we can only test whether a certain helix conformation and orientation is compatible with the NMR data. In fact, the four constraints from the L-CF₃-Phg labels alone would also be compatible with a distorted helix that is slightly tilted away from a transmembrane orientation (see Supplementary Material). However, the independent additional constraints (1x ¹⁵N-Gly, 4x D-CF₃-Phg) supporting the oblique tilt of the central helical segment of PGLa make any alternative interpretation of the data very unlikely.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS SUPPLEMENTARY MATERIAL ACKNOWLEDGEMENTS REFERENCES

 
The tilted T-state of PGLa exhibits quite unexpected properties that do not fit well into the currently discussed structural models of antimicrobial action (Shai, 1999; Matsuzaki, 1999; Huang, 2000; Zasloff, 2002; Strandberg and Ulrich, 2004). It is clearly not compatible with a barrel-stave pore, and the rotational diffusion of the peptide around the membrane normal does not support the carpet model either. The oblique tilt with an order parameter nearly identical to that of the S-state is also not compatible with a toroidal wormhole. Although the high peptide concentration of the T-state may appear to correlate with the threshold for pore formation (Wieprecht et al., 2000), there is no evidence from our experiments that this state should be directly responsible for the destruction of the membrane barrier. Dimerization of PGLa at the membrane surface is a plausible hypothesis to explain our data in light of observations from similar amphiphilic helical membrane peptides from the same family. The T-state may thus be an intermediate in the formation of pores on the way from the monomeric S-state toward a cooperative I-state.

	SUPPLEMENTARY MATERIAL

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An online supplement to this article can be found by visiting BJ Online at http://www.biophysj.org.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS SUPPLEMENTARY MATERIAL ACKNOWLEDGEMENTS REFERENCES

 
CD experiments, ¹⁹F-NMR experiments with D-CF₃-Phg labeled peptides, and effect of structural variation on peptide orientation are available as supporting material in PDF format; the orientational calculation of and is available as MATHEMATICA notebook.

This work was supported by Sonderforschungsbereich 604 (TP B6) and by the Center for Functional Nanostructures of the Deutsche Forschungsgemeinschaft.

Submitted on November 17, 2004; accepted for publication January 27, 2005.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS SUPPLEMENTARY MATERIAL ACKNOWLEDGEMENTS REFERENCES

 
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