TOAC Spin Labels in the Backbone of Alamethicin: EPR Studies in Lipid Membranes

By Marsh, Derek; Jost, Micha; Peggion, Cristina; Toniolo, Claudio

ABSTRACT

Alamethicin is a 19-amino-acid residue hydrophobic peptide that produces voltage-dependent ion channels in membranes. Analogues of the Glu(OMe)^sup 7,18,19^ variant of alamethicin F50/5 that are rigidly spin-labeled in the peptide backbone have been synthesized by replacing residue 1, 8, or 16 with 2,2,6,6-tetramethyl- piperidine-1-oxyl-4-amino-4-carboxyl (TOAC), a helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance spectra are used to determine the insertion and orientation of the TOAC^sup n^ alamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic ^sup 14^N-hyperfine couplings indicate that TOAC^sup 8^ and TOAC^sup 16^ are situated in the hydrophobic core of the membrane, whereas the TOAC^sup 1^ label resides closer to the membrane surface. Anisotropic hyperfine splittings show that alamethicin is highly ordered in the fluid membranes. Experiments with aligned membranes demonstrate that the principal diffusion axis lies close to the membrane normal, corresponding to a transmembrane orientation. Combination of data from the three spin-labeled positions yields both the dynamic order parameter of the peptide backbone and the intramolecular orientations of the TOAC groups. The latter are compared with x-ray diffraction results from alamethicin crystals. Saturation transfer electron paramagnetic resonance, which is sensitive to microsecond rotational motion, reveals that overall rotation of alamethicin is fast in fluid membranes, with effective correlation times <30 ns. Thus, alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from transient or voltage-induced associations.

Abbreviations used: Ac, acetyl: Aib, α-aminoisobutyric acid; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; EDTA, N,N,N’,N’,- ethylenediaminetetraacetic acid; EPR, electron paramagnetic resonance; Hepes, N-(2-hydroxyethyl)piperazine-N’-2-ethanesulphonic acid; NHtBu, tertbutylamino; OMe, methoxy; Phol, phenylalaninol; ST- EPR, saturation transfer EPR; TOAC, 2,2,4,4-tertramethylpiperidine- 1-oxy-4-amino-4-carboxylic acid; V^sub 1^, first-harmonic absorption EPR spectrum detected in phase with respect to the static magnetic held modulation; V^sub 2^’, second-harmonic absorption EPR spectrum detected 90 oul-of-phase with respect to the static magnetic field modulation; Z, benzyloxycarbonyl.

2007 by the Biophysical Society

0006-3495/07/01/473/09 $2.00

INTRODUCTION

Alamethicin is a 19-amino-acid residue peptide from Trichoderma viride (with an N-terminal acetyl and a C-terminal phenylalaninol) that is able to induce voltage-dependent ion conduct ion across lipid membranes (1,2). The channel properties are strongly concentration-dependent and characterized by multiple conduction states (3). Mechanistic studies suggest that consecutive conductance levels are generated by incorporation of single alamethicin molecules into an existing pore aggregate (4). The relative populations of the different conductance levels are responsive to membrane tension and to the intrinsic curvature of the constituent lipids (5,6).

TOAC is a helicogenic nitroxyl amino acid that can be incorporated directly in the backbone of synthetic peptides (7-10). The nitroxide ring is rigidly attached to the C^sup α^-atom of the amino acid and therefore can be used as a spin-label reparler of the orientation and dynamics of the peplide backbone (11). Previous studies have demonstrated the utility of conventional electron paramagnetic resonance (EPR) spectroscopy to determine the location and orientation of TOAC-labeled trichogin GA IV, a membrane-active peptide, in lipid bilayers (12). Such methods exploit both the polarity sensitivity (13) and the angular dependence (14) of the nitroxide EPR spectra.

In this work, we investigate the association of TOAC-labeled alamethicin analogs with phospholipid bilayer membranes by using both conventional and saturation-transfer (ST-) EPR spectroscopy. The TOAC residue is substituted at one of three positions (1, 8, or 16) throughout the sequence of alamethicin. Macroscopically aligned membranes are used to demonstrate that the TOAC-labeled alamethicin assumes a transmembrane orientation, consistent with the relative environmental polarities of the different TOAC positions. Orienlational order parameters for the three TOAC positions allow determination of both the angular amplitude of long-axis motion and the intramolecular tilts of the individual nitroxides. Finally, ST- EPR, which is sensitive to much slower rotational diffusion than conventional EPR (15), and the lack of spin-spin interactions between monomers in the conventional EPR, are used to obtain information on the aggregation slate of the peptide in the membrane.

MATERIALS AND METHODS

Materials

Dimyristoyl phosphatidylcholine (DMPC) was from Avanti Polar Lipids (Alabaster, AL). Spin-labeled derivatives of alamethicin F50/ 5 [TOAC^sup n^, Glu(OMe )^sup 7,18,19^], with n = 1, 8, and 16, were synthesized according to references ( 16,17). The complete amino- acid sequences of the three analogs are:

Ac-TOAC-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-Aib-Val-Aib-Gly-L.eu-AibPro- Val-Aib-Aib-Glu(OMe)-Glu(OMe)-Phol [TOAC1,Glu(OMe)7,18,19]

Ac-Aib-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-TOAC-Val-Aib-Gly-Leu-AibPro- Val-Aib-Aib-Glu(OMe)-Glu(OMe)-Phol [TOAC8,Glu(OMe)7,18,19]

Ac-Aib-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-Aib-Val-Aib-Gly-Leu-Aib- ProVal-TOAC-Aib-Glut(OMe)-Glu(OMe)-Phol[TOAC16,Glu(OMe)7,17,19],

where Aib is α-aminoisobutyric acid and Phol is the β- amino alcohol L-phenylalaninol. Functional measurements demonstrate that the Gln7,18,19 to Glu(OMe)7,18,19 substitution in F50/5 alamethicin does not dramatically reduce the voltage-dependent membrane conductance that is induced by this channel-forming peptide (18).

Sample preparation

DMPC (1 mg) and ~1 mol % of the desired TOAC spin-labeled alamethicin (in MeOH) were codissolved in CH^sub 2^Cl^sub 2^, and the solution then evaporated with dry nitrogen. After keeping under vacuum overnight, the dry mixture was hydrated in 50 l of 10 mM Hepes- (N-(2-hydroxyethyl)piperazine-N’-2ethanesulphonic acid). 10 mM NaCl, 10 m M EDTA (N,N,N’,N’-ethyle nediaminetetraacetc acid). pH 7.8 buffer, with vortex mixing at 37C. The lipid dispersion was then transferred to a 1 mm-diameter glass capillary and pelleted in a benchtop centrifuge. Excess supenialant was removed and the capillaries were flame-sealed.

Aligned planar phospholipid bilayers were formed by evaporating the CH^sub 2^Cl^sub 2^ solution of DMPC plus 1 mol % of TOAC- alamethicin onto the internal faces of a quartz flat cell (Wilmad model No. WG-812. Wilmad-LabGlass, Buena, NJ) by using a stream of dry nitrogen. Residual solvent was removed under vacuum overnight. The oriented lipid film was hydrated with excess buffer containing 150 mM NaCl, at room temperalure. The cells were drained and sealed immediately before measurement, with sufficient buffer retained to ensure compleie hydralion throughout the experiment.

EPR spectroscopy

EPR specrtra were recorded on a Varian Century-Line 9-GHz specrrometer (Varian, Palu Alto, CA) with 100 KHz field modulation. Sample capillaries were accommodated in standard quartz EPR lubes that contained light silicone oil for thermal stability. Temperature was regulated by thermostated nitrogen gas-flow through a quartz Dewar, and was measured with a line-wire thermocouple situated in the silicone oil at the top of the microwave cavity. Samples of ~5- mm height were centered in the rectangular TE^sub 102^ resonator, to minimize microwave- and modulation-field inhomogeneities (19). The microwave H^sub 1^-field at the sample was measured as described in the latter reference. Conventional EPR spectra were recorded in the in-phase first-harmonic absorption mode (V^sub 1^-display), and saturation transfer (ST-) EPR spectra in the out-of-phase second- harmonic absorption mode (V^sub 2^’-display) (20). Oriented bilayer spectra were obtained with the quartz flat cells in a TE^sub 102^ rectangular microwave cavity mounted with its H^sub 1^-field axis horizontal. The entire cavity assembly was thermostated with nitrogen gas-flow.

TOAC orientation

The cristal structure of the ^sup 6^T^sub 2^ twist-boat conformer of TOAC was taken from molecule B of Z-TOAC-(L-Ala)^sub 2^-NHtBu (29). which was obtained from the Cambridge Crystiallographic Data Centre (CCDC code: 123753). In the available crystal structures of α-helical TOAC peptides. ^sup 6^T^sub 2^ is by far the most prevalent conformer of TOAC (30). The crystal structure of native alamelhicin (31) was obtained from the Research Collaboration for Structural Bioinformatics protein database (32) (PDB code: 1 amt). The TOAC residue was substituted for the Aib residue at position 1, 8, or 16 in alamethicin by constraining the transformed coordinates of the TOAC N. C^sup α^ and C’ atoms to coincide with those in alamefhicin. by using nonlinear least-squares optimization.

The orientation φ^sub z^ of the nitroxide z axis of TOAC to the alamethicin molecular axis was determined as described in Marsh (30). The molecular axis of alamethicin was taken as the axis of the longer (N-terminal) helical section. The latter wa\s defined us the line equidistant from the C^sup α^ atoms of residues 4-14. for which the mean radial distance is 2.38 . by nonlinear least-square s fitting. The vector connecting the C^sup α^ atoms of residues 1 and 19 was also used as an alternative definition of the molecular axis. The unpublished structure of [TOAC^sup 16^, Glu(OMe)7,18,19]- alamethicin (33), and variants in which the TOAC residue from position 16 was substituted for the Aib^sup 1^ or Aib^sup 8^ residue, were used in an analogous manner to obtain the orientation of the TOAC nitroxyl axes. This alamethicin analog has the ^sup 6^T^sub 2^ twist-boat conformer of TOAC that is found in the Z-TOAC- (L-Ala)^sub 2^-NHtBu reference peplide.

RESULTS AND DISCUSSION

Conventional spin-label EPR spectra

Fig. 1 shows the EPR spectra of the three different TOAC^sup 1^, TOAC^sup 8^, and TOAC^sup 16^ analogs of [Glu(OMe)7,18,19] alamethicin in DMPC bilayer membranes. Spectra are shown at various temperatures, both above and below the lipid chain-melting temperature of 23C. In the gel phase, the spectra are indicative of strong immobilization on the nanosecond time-scale, but that of the TOAC1 derivative also evidences strong spin-spin broadening. The latter is seen most clearly at 10C as a strong distortion of the baseline by a very broad underlying component (dotted-line spectrum). This spin-spin interaction is caused by aggregation of the spin-labeled alamethicin in the gel-phase membrane. The sharp features in the low-temperature spectra most probably arise from a population of noninteracting spin labels, and therefore indicate some heterogeneity in the degree of aggregation. The aggregation observed by spin-spin interaction in the gel-phase correlates well with functional studies on the effects of the lipid chain-melting transition. The transmemhrane current density mediated by alamethicin in unsupported bilayers of 1-stearoyl-3- myristoylphosphatidylcholine was found to increase dramatically cm entering the gel phase of the membrane from the fluid phase (34). The current density in the gel phase at 24C corresponded to a pore concentration of ~ 10^sup 6^ pores/cm^sup 2^ and decreased to a low level representing only 1 pore/cm^sup 2^ in the fluid phase. Increased pore density implies an increased degree of peptide aggregation and an increased local concentration of alamethicin in the gel phase that manifests itself here as an increase in spin- spin interactions.

Spin-spin broadening is absent from the EPR spectra in the fluid phase at 1 mol % spin label. This rinding indicates that the spin- labeled alamethicin is randomly dispersed in the lipid membrane at temperatures above the lipid chainmelting transition. Similar conclusions have been reached from EPR studies on alamethicin with a flexible spin label at the N- or C-terminus (35.36). The spectra from the three TOAC derivatives arc still highly anisotropic. indicating high ordering or limited motion, in the fluid phase. The extent of spectral anisolropy decreases with increasing temperature. At higher temperatures, the spectra display axial motional averaging, as indicated by the well-defined outer and inner hyperfine splittings. 2A^sub max^ and 2A^sub min^, respectively (see, e.g., (37)).

FIGURE I Conventional EFR spectra (V^sub 1^-display) of [Glu(OMc)7,18,19] alamethicin analogs with TOAC substituted for (A) residue 1. TOAC^sup 1^: (B) residue 8, TOAC8; and (C) residue 16, TOAC16, in DMPC bilayers at the temperatures indicated. (Solid lines, total scan width – 100 Gauss; dotted lines, total scan width: 160 Gauss.)

Temperature dependence

Fig. 2 shows the temperature dependence of the outer hypertine spliiting, 2A^sub max^ for alamethicin with the three different positions of TOAC labeling in DMPC membranes. For the TOAC8 and TOAC16 derivatives, there is a small but abrupt decrease in the value of A^sub max^ at the DMPC chain-melting transition. This decrease in A^sub max^ corresponds to an increase in rotational dynamics of alamethicin on lipid chain fluidizalion. The apparent increase in A^sub max^ for the TOAC1 derivative at 23C most probably is an artifact arising from the spin-spin broadening of this particular label in the gel phase. The values of A^sub max^ at temperatures immediately above the lipid transition are still indicative of a high degree of order, or limited amplitude of angular motion, of the spin-labeled alamethicin. There are, nonetheless, differences between the values of A^sub max^ and their rate of change with temperature for the three different label positions. For each label, the values of A^sup max^ decrease steadily in response to the increased extent of lipid chain motion with increasing temperature.

Isotropic hyperfine couplings

Fig. 3 gives the temperature dependence of the effective isotropic hyperlinc couplings, a^sub o^, defined by Eq. 3, for the different positions of TOAC labeling. The true value of

FIGURE 2 Temperature dependence of the outer hyperfine splitting constant, A^sub max^, for [Glu(OMe)7,18,19] alamethicin TOAC1 (squares), TOAC8 (circles), and TOAC16 (triangles) analogs in DMPC bilayers.

FIGURE 3 Temperature dependence of the effective isotropic hyperfine couplings, a^sub o^, for [Glu(OMe)7,18,19] alamethicin TOAC1 (squares), TOAC8 (circles), and TOAC16 (triangles) analogs in DMPC bilayers.

Orientational order parameters

Fig. 4 gives the temperature dependence of the order parameters. S^sub zz^, for the three TOAC analogs of alamethicin in DMPC bilayer membranes in the fluid phase. According to the criterion of constant a^sub o^, the data in Fig. 3 suggest that motional narrowing theory should he applicable above 55C for all except the TOAC16 analog. As already noted, the spectra are axially symmetric in this temperature regime (see Fig. 1). The values given in Fig. 4 therefore should be reasonably representative of the time-average angular amplitude of the spin-label z axis, relative to the director for the uniaxial rotational motion. At temperatures below those for which data is given in Fig. 4, motional narrowing theory can no longer be relied upon for determining order parameters.

Fig. 5 shows the conventional EPR spectra of the three TOAC- containing analogs of alamethicin in aligned multi-bilayers of fully hydrated DMPC in the fluid phase. Spectra are shown for the static magnetic field parallel (solid lines) and perpendicular (dashed lines) to the normal to the orienting quartz substrate on which the multibilayers are deposited. Although the degree of alignment of the sample is probably not completely homogeneous, and at these temperatures (30-32C) the rotational motion is not yet in the last regime (especially for the TOAC16 analog), there is clear anisotropy between spectra recorded with the parallel and perpendicular orientations of the magnetic field. For all three TOAC positions, the largest hyperfine splitting of the first derivative-like single absorption lines is obtained in the parallel orientation with the magnetic field lying along the substrate normal (see, e.g., (14)). This finding confirms that the director, N, for the uniaxial rotation lies along the membrane normal, consistent with a transmembrane orientation of the alamethicin molecule as indicated by the isotropic hyperfine couplings.

FIGURE 4 Temperature dependence of the effective order parameters, S^sub zz^, for [Glu(OMe)7,18,19 alamethicin TOAC1 (squares), TOAC8 (circles), and TOAC16 (triangles) analogs in DMPC bilayers. Solid lines are experimental measurements; dotted lines are a nonlinear least-squares fit of Eq. 6 to the temperature dependence, with constant θ, for each TOAC position (see text and Fig. 6).

FIGURE 5 Conventional V^sub 1^-EPR spectra of [TOAC11, Glu(OMe)7,18,19] alamethicin analogs in aligned DMPC bilayers. (Solid lines, magnetic field parallel to the membrane normal; dashed lines, magnetic field perpendicular to the membrane normal.) (A) TOAC1-alamethicin at 30C; (B) TOAC8-alamethicin at32C; and (C) TOAC16-alamethicin at 32C. Total scan width = (100) Gauss.

FIGURE 6 Orientation of TOAC-labeled alamethicin in a lipid membrane. The principal molecular diffusion axis, R, is inclined at instantaneous angle γ to the membrane normal N. The nitroxide z axis is oriented at constant angle θ^sub z^ to R. The experimental order parameter, S^sub zz^, of the nitroxide z axis is given by Eq. 6, where, for axial symmetry, [left angle bracket]P^sub 2^(cosγ)[right angle bracket] is the order parameter of the alamethicin diffusion axis.

The dotted lines in Fig. 4 represent a nonlinear least-squares tit of Eq. 6 to the temperature dependence of S^sub zz^ for all three TOAC labels, under the assumption that the spin-label inclination to the diffusion axis is temperature independent. The order parameter of the diffusion axis, relative to N, then varies from [left angle bracket]P^sub 2^(cosγ)[right angle bracket] = 0.87-0.70 over the temperature range 60-85C. The local orientation of the individual spin labels is characterized by the fixed values θ^sub z^ = 30, 25, and 20 for TOAC1, TOAC8, and TOAC16, respectively. Judging from the goodness of the fits in Fig. 4, only for TOAC1 are there significant changes in θ^sub z^ with temperature, possibly corresponding to a local unwinding of the helix or other conformational reorientation at the first residue position.

TOAC orientation in alamethicin

Fig. 7 shows one of the molecules (A) in the crystal structure of native alamethicin (31) into which the crystal structure of the TOAC moiety from molecule B of Z-TOAC-(L-Ala)2-NHtBu (29) has been incorporated at residue position 1, 8, or 16. This was done by constraining the coordinates of the TOAC N, Cα, and C’ atoms to coincide with those of Aib1, Aib8, or Aib16 in alamethicin. If the axis of the longer helical segment is defined as the line that is equidistant from the Cα atoms of residues 4-14, the inclination of the nitroxide z axis to this axis is θ^sub α^ = 7, 15. and 34, for TOAC at residue positions 1, 8, and 16, respectively. For the recently solved structure of [TOAC16, Glu(OMe)7,18,19) alamelhicin (33), the orientation of the spin-label z axis to the longer helical axis is θ^sub α^ = 10-12 and, for this TOAC structure grafted at residue positions 1 and 8, is θ^sub α^ = 4-7 and 8-9, respectively. In terms of residue position, these values for the TOAC orientation θ^sub α^ are in the opposite order to those of θ^sub z^ that are derived from the EPR results. From this, one must conclude that the diffusion axis does not coincide with the helical axis between residues 4 and 14, as defined above. Taking the more recent crystal structure, the rotation axis R is tilted relative to the principal helix axis by ~30. This value may be somewhat of an upper estimate because of the effects of local helix distortions that were referred to above. Note that taking the mirror-image ^sup 2^T^sub 6^ twist-boat conformer of TOAC would predict nitroxide z-axis orientations that are incompatible with the EPR order-parameter measurements (30,44).

FIGURE 7 Crystal structure for molecule A of native alamethicin ((31); PDB: 1amt) with the TOAC structure from molecule B of Z-TOAC- (L-Ala)2-NHtBu ((29) CCDC: 123753) substituted for Aib at residue position 1, 8, or 16. The alamethicin molecule is oriented relative to the membrane surfaces as predicted in the OPM database (53).

Saturation transfer spin-label EPR spectra

Fig. 8 shows me saturation transfer EPR spectra of the three different TOAC1, TOAC8, and TOAC16 analogs of [Glu(OMe)7,18,19] alamethicin in DMPC bilayer membranes. Spectra are shown at various temperatures, both above and below the lipid chain-melting temperature of 23C. The spectra are scaled to line height, rather than to the absolute intensity, which decreases with increasing temperature. In the gel phase, the ST-EPR spectra have appreciable intensity. not only in the overall spectrum, but also in the diagnostic regions at intermediate positions in the low-, high-, and center-field manifolds of the ^sup 14^N-hyperfine structure. These nonvanishing ST-EPR intensities reflect the response of the peptide to the extremely slow rotational diffusion of the gel-phase lipids, with effective correlation times beyond the microsecond regime (45,46). Immediately above the lipid chain-melting transition, the overall intensity of the ST-EPR spectrum drops abruptly and the spectral lineshape changes because of preferential reduction in the line heights at the diagnostic L”, C’, and H” positions relative to the stationary turning points at positions L, C and H, respectively (see, e.g., (15)). These spectra are characteristic of very fast motion, no longer in the microsecond regime (at least for the central C’ region of the spectrum), and reflect the response of the peptide mobility to the rapid lipid chain motions in the fluid membrane phase (45,47).

Fig. 9 gives the temperature dependence of the normalized ST- intensily, I^sub ST^, and the diagnostic line-height ratios, L”/L, C’/C, and H”/H, for the TOAC8 alamethicin analog in DMPC membranes. This is the analog with highest ST-EPR intensities and, therefore, that most likely to exhibit any microsecond motions that are associated with the whole peptide. All four ST-EPR parameters clearly reflect the change in overall peptide dynamics at the gel- fluid phase transition, which occurs at ~23C. The values of the C’/ C line-height ratio and of the ST integral are very low in the fluid phase, beyond those for which ST-EPR calibrations were made. This corresponds to an effective rotational correlation time of <2.9 10^sup -8^ s (28). In addition, the L''/L ratio is seen to increase with increasing temperature, i.e., with decreasing correlation time. This is a feature of incipient motional narrowing in ST-EPR spectra (15), that again is consistent with correlation times of <10^sup - 7^ s. Such rapid rotational reorientation suggests rather strongly that the peptide is not aggregated in fluid DMPC membranes, as was concluded already from the lack of spin-spin broadening of the conventional EPR spectra. Standard hydrodynamic theory (see, e.g., (48)) predicts a rotational correlation time of 1.4-2.8 10^sup -7^ s for a single transmembrane α-helix in a membrane of effective viscosity 2.5-5 P (49). For a dimer, this value increases to 3.5- 7.0 10^sup -7^ s, and for a tetramer to 5.6-11.3 10^sup -7^ s. It therefore seems most likely that the TOAC alamethicin analogs are monomeric (at a concentration of 1 mol %) in fluid DMPC bilayers, as suggested previously tor alamethicin in vesicles of unsaturated phosphatidylcholines (35,36). Note that substitution of Gln residues, particularly the conserved Gln7, by Glu(OMe) might reduce somewhat the propensity of alamethicin to form pores, as suggested by the lower conductance (18).

FIGURE 8 Saturation transfer EPR spectra (V^sub 2^’-display) of [Glu(OMe)7,18,19] alamethicin analogs with TOAC substituted for (A) residue 1, TOAC1; (B) residue 8, TOAC8; and (C) residue 16, TOAC16 in DMPC bilayers at the temperatures indicated. Total scan width = 160 Gauss.

FIGURE 9 Temperature dependence of the integrated intensity, I^sub ST^ (open circles), and diagnostic line-height ratios, L”/L (squares), C’/C (solid circles), and H”/H (triangles), from the ST- EPR spectra of [TOAC8, Glu(OMe)7,18,19] alamethicin in DMPC bilayers.

CONCLUSIONS

The results from aligned samples and from the relative polarities of the environments of the different TOAC positions demonstrate that Glu(OMe)7,18,19 alamethicin adopts a transmembrane orientation in fluid bilayer membranes of DMPC. This is in agreement with other spectroscopic studies on unmodified and chemically labeled alamethicins (43,50). Certain models for the induction of ion channels propose a switching of the alamethicin long-axis from a surface to a transmembrane orientation (see, e.g., (2)). The present results show that the bulk of the peptide has a transmembrane orientation and therefore such channels are most likely formed by self-assembly within the membrane.

The combined order parameter measurements from the different TOAC positions indicate that the tilt of the long axis of the peptide, relative to the membrane normal, is fairly small with values of [left angle bracket]P^sub 2^(cosγ)[right angle bracket] corresponding to effective tilt angles of 17-27 over the temperature range 60-85C. It is expected that the tilt of alamethicin is restricted because the length of the molecule (~29 from Cα of residue 1 to C^sup α^ of residue 19) is relatively short compared with the thickness of a DMPC bilayer. For the latter, the hydrophobic thickness is ~26 and the total thickness is ~37 at 30C, which extrapolate to 21 and 30 , respectively, at 85C using an expansion coefficient of -0.004 per degree (51). Orientation of alamethicin according to the distribution of polarily/ hydrophobicity in the molecule, as reported in the OPM database (see Fig. 7), predicts a transmembrane alignment of alamethicin with a hydrophobic depth of 28 and a lilt of 16 8 (52,53). This theoretical prediction is therefore essentially in accord with the present experimental measurements.

An interesting feature of the angular motion of the TOAC spin labels, relative to that of spin-labeled lipid chains (see, e.g., (54)), is that the rotational diffusion is slow on the EPR timescale (~ns) in fluid membranes, except at rather high temperatures (>60C). This reflects the rigidity of the helical backbone of alamethicin and the anchoring of the TOAC ring at the C^sup α^-position of the helix.

On the longer (s) timescale of ST-EPR, however, rotation about the long axis of alamethicin is relatively rapid. This means that alamethicin is not funning large pore aggregates, which would have \rotational correlation times in the micro-second regime. Most likely, the bulk of the alamethicin is monomeric in the fluid membrane, and pore formation (and growth) occurs via transient association of the monomeric species. This is in accordance with the electrophysiological channel behavior and proposals from other spectroscopic studies (43).

We thank Frau B. Angerstein and Frau B. Freyberg for skillful technical assistance, Dr. Marco Crisma for providing the coordinates of [TOAC16, Glu(OMe)7,18,19] alamethicin, and Frau I. Dreger for help with preparing Fig. 7.

doi: 10.1529/biophysj.106.092775

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20. Hemminga, M. A., P. A. De Jager, D. Marsh, and P. Fajer. 1984. Standard conditions for the measurement of saturation transfer ESR spectra. J. Magn. Reson. 59:160-163.

21. Rama Krishna, Y. V. S., and D. Marsh. 1940. Spin label ESR and ^sup 31^P-NMR studies of the cubic and inverted hexagonal phases of dimyristoylphosphatidylcholine/myristic acid (1:2, mol/mol) mixtures. Biochem. Biophys. Acta. 1024:89-94.

22. Schorn, K., and D. Marsh, 1996. Lipid chain dynamics and molecular location of diacylglycerol in hydrated binary mixtures with phosphatidylcholine: spin label ESR studies. Biochemistry. 35:3831-3836.

23. Schorn, K., and D, Marsh. 1997. Extracting order parameters from powder EPR lineshapes for spin-labeled lipids in membranes. Spectrochim. Acta [A]. 53:2235-2240.

24. Ondar, M. A., O. Ya. Grinberg, A. A. Dubinskii, and Ya. S. Lebedev. 1985. Study of the effect of the medium on the magnetic- resonance parameters of nitroxyl radicals by high-resolution HPR spectroscopy. Sov. J. Chem. Phys. 3:781-792.

25. Horvth. L. I., and D. Marsh. 1983. Analysis of multicomponent saturation transfer ESR spectra using the integral method: application to membrane systems. J. Magu. Reson. 54:363-373.

26. Marsh, D., and L. I. Horvth. 1992. A simple analytical treatment of the sensitivity of saturation transfer EPR spectra to slow rotational diffusion. J. Magn. Reson. 99:323-331.

27. Marsh, D. 1999. Spin label ESR spectroscopy and FTIR spectroscopy for structural/dynamic measurements on ion channels. Methods Enzymol. 294:59-92.

28. Horvth, L. I., and D. Marsh. 1988. Improved numerical evaluation of saturation transfer electron spin resonance spectra. J. Magn. Reson. 80: 314-317.

29. Flippen-Anderson, J. L., C. George, G. Valle, E. Valente, A. Bianco, F. Formaggio, M. Crisma, and C. Toniolo. 1996. Crystallographic characterization of geometry and conformation of TOAC, a nitroxide spin-labeled C^sub α,α^-disubstituted glycine, in simple derivatives and model peptides. Int. J. Pept. Protein Res. 47:231-239.

30. Marsh, D. 2006. Orientation of TOAC amino-acid spin labels in α-helices and β-strands. J. Magn. Reson. 180:305-310.

31. Fox, R. O., Jr., and F. M. Richards. 1982. A voltage-gated ion channel model inferred from the crystal structure of alamethicin at 1.5 resolution. Nature. 300:325-330.

32. Herman, H. M., J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov, and P. E. Bourne, 2000. The protein data bank. Nucleic Acids Res. 28:235-242.

33. Crisma, M., F, Formaggio, M. Jost, C. Peggion, and C. Toniolo. 2005. Crystal structure of a spin-labeled alamethicin analogue. In 1st European Conference on Chemistry for Life Sciences: Understanding the Chemical Mechanisms of Life. Book of Abstracts, DCSB 234. Rimini, Italy.

34. Boheim, G., W. Hanke, and H, Eibl. 1980. Lipid phase transition in planar bilayer membrane and its effect on carrier- and pore-mediated ion transport. Proc Natl. Acad. Sci. USA. 77:3403- 3407.

35. Archer, S. J., J. F. Ellena, and D. S. Caliso. 1991. Dynamics and aggregation of the peptide ion channel alamethicin. Biophys. J. 60: 389-398.

36. Barranger-Mathys, M., and D. S. Cafiso, 1994. Collisions between helical peptides in membranes monitored using electron paramagnetic resonance: evidence that alamethicin is monomeric in the absence of a membrane potential. Biophys. J. 67:172-176.

37. Marsh, D. 1981. Electron spin resonance: spin labels. In Membrane Spectroscopy. Molecular Biology, Biochemistry and Biophysics, Vol. 31. E. Grell, editor. Springer-Verlag, Berlin, Heidelberg, New York. 51-142.

38. Marsh, D. 2002. Membrane water-penetration profiles from spin labels. Eur. Biophys. J. 31:559-562.

39. Marsh, D. 2002. Polarity contributions to hyperfine splittings of hydrogen-bonded nitroxides-the microenvironment of spin labels. J. Magn. Reson. 157:114-118.

40. Schreier, S., S. R. Barbosa, F. Casallanovo, R. Vieira, E. M. Cilli, A. C. M. Paiva, and C. R. Nakaie. 2004. Conformational basis for the biological activity of TOAC-labeled angiotensin II and bradykinin: electron paramagnetic resonance, circular dichroism, and fluorescence studies. Biopolymers. 74:389-402.

41. Monaco, V., F. Formaggio, M. Crisma, C. Tomolo, P. Hanson, and G. Millhauser. 1999. Orientation and immersion depth of a helical lipopeptaibol in membranes using TOAC as an ESR probe. Biopolymers. 50:239-253.

42. Fernandez, R, M., R. F. F. Vieira, C. R. Nakaie, M. T. Lamy, and A. S. Ito. 2005. Acid-base titration of melanocortin peptides: evidence of Trp rotational conformer interconversion. Biopolym. Pept. Sci. 80:643-650.

43. Barranger-Mathys, M., and D. S. Cafiso. 1996. Membrane structure of voltage-gated channel forming peptides by site- directed spin-labeling. Biochemistry. 35:498-505.

44. Hanson, P., D. J. Anderson, G. Martinez, G. Millhauser, F. Formaggio, M. Crisma, C. Toniolo, and C. Vita. 1998. Electron spin resonance and structural analysis of water soluble, alanine-rich peptides incorporating TOAC. Mol. Phys. 95:957-966.

45. Marsh, D. 1980. Molecular motion in phospholipid bilayers in the gel phase: long axis rotation. Biochemistry. 19:1632-1637.

46. Fajer, P., A. Watts, and D. Marsh. 1992. Saturation transfer, continuous wave saturation, and saturation recovery electron spin resonance studies of chain-spin labeled phosphatidylcholines in the low temperature phases of dipalmitoyl phosphatidylcholine bilayers. Effects of rotational dynamics and spin-spin interactions. Biophys J. 61:879-891.

47. Bartucci, R., T. Pli, and D. Marsh. 1993. Lipid chain motion in an interdigitated gel phase: conventional and saturation transfer ESR of spin-labeled lipids in dipalmitoylphosphalidylc\haline- glycerol dispersions. Biochemistry. 32:274-281.

48. Marsh, D., and L. I. Horvth. 1989. Spin-label studies of the structure and dynamics of lipids and proteins in membranes. In Advanced EPR. Applications in Biology and Biochemistry. A. J. Hoff. editor. Elsevier, Amsterdam, The Netherlands. 707-752.

49. Cherry, R. J., and R. E. Godfrey. 1981. Anisotropic rotation of bacteriorhodopsin in lipid membranes. Biophys. J. 36:257-276.

50. Vopel, H. 1987. Comparison of the conformation and orientation of alamethicin and melittin in lipid membranes. Biochemistry. 26: 4652-4672.

51. Nagle, J. F., and S. Tristram-Nagle. 2000. Structure of lipid bilayers. Biochim. Biophys. Acta. 1469:159-195.

52. Lomize, A. L., I. D. Pogozheva, M. A. Lomize, and H. I. Mosberg. 2006. Positioning of proteins in membranes: a computational approach. Protein Sci. 15:13I8-1333.

53. Lomize, M. A., A. L. Lomize, I. Pogozheva, and H. I. Mosberg. 2006. OPM: orientations of proteins in membranes database. Bioinformatics. 22:623-625.

54. Schorn, K., and D. Marsh. 1996. Lipid chain dynamics in diacylglycerolphosphatidycholine mixtures studied by slow-motional simulations of spin label ESR spectra. Chem. Phys. Lipids. 82:7-14.

Derek Marsh,* Micha Jost,[dagger] Cristina Peggion,[dagger] and Claudio Toniolo[dagger]

* Max-Planck-Institut fr biophysikalische Chemie, Abteilung Spektroskopie, Gttingen, Germany; and [dagger] Department of Chemistry, University of Padova, Padova, Italy

Submitted July 4, 2006, and accepted for publication September 14, 2006.

Address reprint requests to Dr. Derek Marsh, Tel.: 44-551-201- 1285; E-mail: dmarsh@gwdg.de.

Copyright Biophysical Society Jan 15, 2007

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