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Interactions Involved in the Realignment of Membrane-associated Helices

2006; Elsevier BV; Volume: 281; Issue: 12 Linguagem: Inglês

10.1074/jbc.m513151200

1083-351X

Christopher Aisenbrey, R. Kinder, Erik Goormaghtigh, Jean‐Marie Ruysschaert, Burkhard Bechinger,

Lipid Membrane Structure and Behavior

A series of histidine-containing peptides (LAH4X6) was designed to investigate the membrane interactions of selected side chains. To this purpose, their pH-dependent transitions from in-plane to transmembrane orientations were investigated by attenuated total reflection Fourier transform infrared and oriented solid-state NMR spectroscopies. Peptides of the same family have previously been shown to exhibit antibiotic and DNA transfection activities. Solution NMR spectroscopy indicates that these peptides form amphipathic helical structures in membrane environments, and the technique was also used to characterize the pK values of all histidines in the presence of detergent micelles. Whereas one face of the amphipathic helix is clearly hydrophobic, the opposite side is flanked by four histidines surrounding six leucine, alanine, glycine, tryptophan, or tyrosine residues, respectively. This diversity in peptide composition causes pronounced shifts in the midpoint pH of the in-plane to transmembrane helical transition, which is completely abolished for the peptides carrying the most hydrophilic amino acid residues. These properties open up a conceptually new approach to study in a quantitative manner the hydrophobic as well as specific interactions of amino acids in membranes. Notably, the resulting scale for whole residue transitions from the bilayer interface to the hydrophobic membrane interior is obtained from extended helical sequences in lipid bilayers. A series of histidine-containing peptides (LAH4X6) was designed to investigate the membrane interactions of selected side chains. To this purpose, their pH-dependent transitions from in-plane to transmembrane orientations were investigated by attenuated total reflection Fourier transform infrared and oriented solid-state NMR spectroscopies. Peptides of the same family have previously been shown to exhibit antibiotic and DNA transfection activities. Solution NMR spectroscopy indicates that these peptides form amphipathic helical structures in membrane environments, and the technique was also used to characterize the pK values of all histidines in the presence of detergent micelles. Whereas one face of the amphipathic helix is clearly hydrophobic, the opposite side is flanked by four histidines surrounding six leucine, alanine, glycine, tryptophan, or tyrosine residues, respectively. This diversity in peptide composition causes pronounced shifts in the midpoint pH of the in-plane to transmembrane helical transition, which is completely abolished for the peptides carrying the most hydrophilic amino acid residues. These properties open up a conceptually new approach to study in a quantitative manner the hydrophobic as well as specific interactions of amino acids in membranes. Notably, the resulting scale for whole residue transitions from the bilayer interface to the hydrophobic membrane interior is obtained from extended helical sequences in lipid bilayers. To date, high resolution three-dimensional structural information on membrane proteins remains sparse. Therefore, prediction and computational methods that allow identifying membrane-spanning segments from their amino acid sequence remain an important tool to establish a first topological model. To this purpose, a number of "hydrophobicity scales" have been developed and later improved using computational algorithms (1Chen C.P. Rost B. Appl. Bioinform. 2002; 1: 1-15Google Scholar). These are either based on experimental data (2Wimley W.C. White S.H. Nat. Struct. Biol. 1996; 3: 842-848Crossref PubMed Scopus (1385) Google Scholar, 3Hessa T. Kim H. Bihlmaier K. Lundin C. Boekel J. Andersson H. Nilsson I. White S.H. von Heijne G. Nature. 2005; 433: 377-381Crossref PubMed Scopus (783) Google Scholar) or mixed experimental and educated guesses (4von Heijne G. Eur. J. Biochem. 1981; 120: 275-278Crossref PubMed Scopus (110) Google Scholar, 5Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17086) Google Scholar, 6Rees D.C. DeAntonio L. Eisenberg D. Science. 1989; 245: 510-513Crossref PubMed Scopus (275) Google Scholar, 7Engelman D.M. Steitz T.A. Goldman A. Annu. Rev. Biophys. Biophys. Chem. 1986; 15: 321-353Crossref PubMed Scopus (1198) Google Scholar). Furthermore, from the few known three-dimensional structures as well as from biochemical experiments that determine the topology of membrane proteins (8Moller S. Kriventseva E.V. Apweiler R. Bioinformatics. 2000; 16: 1159-1160Crossref PubMed Scopus (90) Google Scholar), knowledge-based statistical scales have been established (9Degli E.M. Crimi M. Venturoli G. Eur. J. Biochem. 1990; 190: 207-219Crossref PubMed Scopus (81) Google Scholar, 10Gromiha M.M. Ponnuswamy P.K. Int. J. Pept. Protein Res. 1996; 48: 452-460Crossref PubMed Scopus (19) Google Scholar, 11Jones D.T. Taylor W.R. Thornton J.M. Biochemistry. 1994; 33: 3038-3049Crossref PubMed Scopus (706) Google Scholar, 12Punta M. Maritan A. Proteins. 2003; 50: 114-121Crossref PubMed Scopus (26) Google Scholar). A good algorithm should reliably identify all transmembrane helices and differentiate membrane-spanning domains from sequences that compose a hydrophobic helix within the interior of a soluble protein (13Promponas V.J. Palaios G.A. Pasquier C.M. Hamodrakas J.S. Hamodrakas S.J. In Silico Biol. 1999; 1: 159-162PubMed Google Scholar, 14Ikeda M. Arai M. Lao D.M. Shimizu T. In Silico Biol. 2002; 2: 19-33PubMed Google Scholar, 15Jayasinghe S. Hristova K. White S.H. J. Mol. Biol. 2001; 312: 927-934Crossref PubMed Scopus (201) Google Scholar, 16Chen C.P. Kernytsky A. Rost B. Protein Sci. 2002; 11: 2774-2791Crossref PubMed Scopus (175) Google Scholar, 17Kall L. Sonnhammer E.L. FEBS Lett. 2002; 532: 415-418Crossref PubMed Scopus (69) Google Scholar). Furthermore, the membrane exhibits very different properties in the interior, at the interface or within the regions directly next to the surface (11Jones D.T. Taylor W.R. Thornton J.M. Biochemistry. 1994; 33: 3038-3049Crossref PubMed Scopus (706) Google Scholar, 18von Heijne G. J. Mol. Biol. 1992; 225: 487-494Crossref PubMed Scopus (1401) Google Scholar, 19Sonnhammer E.L. von Heijne G. Krogh A. Proc. Int. Conf. Intell. Syst. Mol. Biol. 1998; 6: 175-182PubMed Google Scholar, 20Tusnady G.E. Simon I. J. Mol. Biol. 1998; 283: 489-506Crossref PubMed Scopus (946) Google Scholar, 21Hessa T. White S.H. von Heijne G. Science. 2005; 307: 1427Crossref PubMed Scopus (158) Google Scholar). These marked differences in physico-chemical environment are also important for the alignment of polypeptide sequences either parallel or perpendicular to the membrane surface (22Bechinger B. FEBS Lett. 2001; 504: 161-165Crossref PubMed Scopus (23) Google Scholar). How peptides orient relative to the membrane normal is of considerable importance for the activity and regulation of helical sequences such as antibiotic peptides, DNA transfectants, or signal sequences (23Vogt T.C.B. Bechinger B. J. Biol. Chem. 1999; 274: 29115-29121Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 24Kichler A. Leborgne C. März J. Danos O. Bechinger B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1564-1568Crossref PubMed Scopus (196) Google Scholar). Furthermore, biophysical studies indicate that some membrane-inserted proteins may exhibit a more loosely folded structure, thereby resembling a tethered assembly of individual helices. These include some of the colicin channel domains (25Zakharov S.D. Lindeberg M. Griko Y. Salamon Z. Tollin G. Prendergast F.G. Cramer W.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4282-4287Crossref PubMed Scopus (76) Google Scholar) or the antiapoptotic Bcl-xL protein (26Losonczi J.A. Olejniczak E.T. Betz S.F. Harlan J.E. Mack J. Fesik S.W. Biochemistry. 2000; 39: 11024-11033Crossref PubMed Scopus (92) Google Scholar). The interactions of these proteins with the membrane are therefore governed by the same mechanisms as those important for smaller peptide sequences. A reliable prediction of membrane-spanning protein domains requires good knowledge of the free energies (or a parameter that is correlated to that) that are involved when placing amino acid side chains in the hydrophobic interior of the membrane. Bilayer insertion involves the transfer from the aqueous buffer to the membrane interface and then on into the hydrophobic interior. Most hydrophobicity scales therefore monitor the transition from the aqueous to a low dielectric environment (4von Heijne G. Eur. J. Biochem. 1981; 120: 275-278Crossref PubMed Scopus (110) Google Scholar, 5Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17086) Google Scholar, 6Rees D.C. DeAntonio L. Eisenberg D. Science. 1989; 245: 510-513Crossref PubMed Scopus (275) Google Scholar, 7Engelman D.M. Steitz T.A. Goldman A. Annu. Rev. Biophys. Biophys. Chem. 1986; 15: 321-353Crossref PubMed Scopus (1198) Google Scholar). A series of model peptides is presented in this paper that provide experimental access to the free energies associated with the transitions from in-plane to transmembrane helical alignments and thus from residue localizations at the membrane interface to the bilayer interior. The LAH4 peptide (27Bechinger B. J. Mol. Biol. 1996; 263: 768-775Crossref PubMed Scopus (173) Google Scholar), which was used as a design template, exhibits pronounced antimicrobial activity (23Vogt T.C.B. Bechinger B. J. Biol. Chem. 1999; 274: 29115-29121Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) and functions as a potent DNA transfectant (24Kichler A. Leborgne C. März J. Danos O. Bechinger B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1564-1568Crossref PubMed Scopus (196) Google Scholar). Interestingly, "mutagenesis" experiments indicate that this latter activity is strongly dependent on the capacity of the peptide to change its alignment relative to the membrane normal (24Kichler A. Leborgne C. März J. Danos O. Bechinger B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1564-1568Crossref PubMed Scopus (196) Google Scholar, 28Kichler A. Bechinger B. Danos O. Med. Sci. 2003; 19: 1046-1047Google Scholar). Here we investigate in a more systematic manner how the amino acid composition of LAH4-type peptides can be used to modulate the transition pH. The LAH4-derived sequences are composed of four histidines interrupted by a hydrophobic stretch of alanines and leucines. Furthermore, several lysines at each terminus act as membrane anchors and increase the solubility of the peptides in polar solvents. Whereas the central core of the peptide sequence is sufficiently long and hydrophobic to be able to span the lipid bilayer, the histidines are arranged in such a manner as to allow the formation of an amphipathic α-helical structure (Fig. 1). The histidine side chains exhibit pKa values close to 6.0 when in an aqueous environment, thus being positively charged at acidic pH and polar but uncharged at pH ≥7 (Table 1). Histidines have been used previously to control the membrane interactions of model peptides (27Bechinger B. J. Mol. Biol. 1996; 263: 768-775Crossref PubMed Scopus (173) Google Scholar, 29Ladokhin A.S. White S.H. Biochemistry. 2004; 43: 5782-5791Crossref PubMed Scopus (80) Google Scholar).TABLE 1pK and c values of the histidine residues in LAH4X6 peptides in the presence of 386 mm DPC (see "Results" for details)XpK valuesc valuesGly6.0, 6.1, 6.1, 6.10.71, 0.64, 0.74, 0.92Ala5.9, 6.0, 5.9, 6.00.63, 0.85, 0.73, 0.85Leu5.8, 5.8, 5.9, 5.80.85, 0.77, 0.72, 0.86 Open table in a new tab The helical wheel diagram of LAH4X6 indicates how six amino acids of variable composition (labeled X) are interspersed between the histidines (Fig. 1). In this design, hydrophobic residues at the X positions exert a strong driving force for membrane insertion. This is counter-acted by the preference of the histidine side chains to remain in the water phase, an interaction that is strongly dependent on the pH of the surrounding medium. Therefore, the transmembrane insertion of the peptides is a function of both the pH of the environment and the "hydrophobicity" of the X amino acids within the amphipathic α-helix. The pH-dependent in-plane to transmembrane transition can thus be used to directly test for the relative hydrophobicity of the X residues. Previously, it has been shown that oriented solid-state NMR or ATR-FTIR 3The abbreviations used are: ATR, attenuated total reflection; FTIR, Fourier transform infrared; Fmoc, N-(9-fluorenyl)methyloxycarbonyl; DPC, dodecyl phosphocholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine; MALDI, matrix-assisted laser desorption ionization; HPLC, high pressure liquid chromatography; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy. 3The abbreviations used are: ATR, attenuated total reflection; FTIR, Fourier transform infrared; Fmoc, N-(9-fluorenyl)methyloxycarbonyl; DPC, dodecyl phosphocholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine; MALDI, matrix-assisted laser desorption ionization; HPLC, high pressure liquid chromatography; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy. spectroscopy can be used to follow the alignment of helical peptides in membranes. The 15N chemical shift (30Bechinger B. Sizun C. Concepts Magn. Reson. 2003; 18A: 130-145Crossref Scopus (102) Google Scholar) and the dichroic ratio (31Bechinger B. Ruysschaert J.M. Goormaghtigh E. Biophys. J. 1999; 76: 552-563Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) provide sensitive indicators of helical tilt angles when α-helices are incorporated in oriented phospholipids bilayers. When the 15N NMR technique is applied to membranes oriented with the normal parallel to the magnetic field direction, the measurement of 15N chemical shifts of 180 ppm agree with transmembrane helix alignments (30Bechinger B. Sizun C. Concepts Magn. Reson. 2003; 18A: 130-145Crossref Scopus (102) Google Scholar, 32Bechinger B. Aisenbrey C. Bertani P. Biochim. Biophys. Acta. 2004; 1666: 190-204Crossref PubMed Scopus (62) Google Scholar). On the other hand, ATR-FTIR spectroscopy of oriented membrane samples allows one to monitor the average peptide alignment and conformation by measurement of the dichroic ratio (33Ivanov D. Dubreuil N. Raussens V. Ruysschaert J.M. Goormaghtigh E. Biophys. J. 2004; 87: 1307-1315Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 34Goormaghtigh E. Raussens V. Ruysschaert J.M. Biochim. Biophys. Acta. 1999; 1422: 105-185Crossref PubMed Scopus (508) Google Scholar, 35Lopes S.C. Goormaghtigh E. Cabral B.J. Castanho M.A. J. Am. Chem. Soc. 2004; 126: 5396-5402Crossref PubMed Scopus (25) Google Scholar) and the frequencies of the amide bands (31Bechinger B. Ruysschaert J.M. Goormaghtigh E. Biophys. J. 1999; 76: 552-563Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 36Goormaghtigh E. Cabiaux V. Ruysschaert J.M. Subcell. Biochem. 1994; 23: 405-450Crossref PubMed Scopus (355) Google Scholar), respectively. The characteristic frequencies are 1662–1645 cm–1 for α-helical peptides, 1689–1682 cm–1 for β-sheet conformations, and 1644–1637 cm–1 in the case of random coil sequences (36Goormaghtigh E. Cabiaux V. Ruysschaert J.M. Subcell. Biochem. 1994; 23: 405-450Crossref PubMed Scopus (355) Google Scholar, 37Goormaghtigh E. Cabiaux V. Ruysschaert J.M. Eur. J. Biochem. 1990; 193: 409-420Crossref PubMed Scopus (461) Google Scholar). Furthermore, the dichroic ratio of the amide I band is a direct indicator of the average tilt angle of helical polypeptides. Whereas in-plane oriented peptides exhibit R values around 1.3, this parameter augments by a factor of 2–3 for transmembrane helix orientations. Here we have used both approaches to investigate several peptides of the LAH4X6 series. In order to analyze the data in quantitative detail, dynamic equilibria between membrane-associated states are considered. The underlying model and theory have been described in detail elsewhere (38Aisenbrey, C. (2003) Étude Topologique de Polypeptides Membranaires par RMN du Solide et Spectroscopie Infrarouge par Réflexion Totale Attenuée. Ph.D. thesis, University Louis Pasteur, Strasbourg, FranceGoogle Scholar). In short, the following membrane-associated states are considered: IPch ↔ IPo ↔ TM. Here IP indicates the in-plane oriented peptide helices, with the histidines charged (ch) or neutral (o), respectively, and TM indicates the transmembrane inserted configuration. Bulk water being absent in these samples, other states, such as peptides dissolved in the aqueous phase, are neglected in this model. Due to the high energy of placing a charged residue in the membrane environment (39Israelachvilli J.N. Marcelja S. Horn R.G. Q. Rev. Biophys. 1980; 13: 121-200Crossref PubMed Scopus (1133) Google Scholar), it is assumed that all four histidines have to discharge prior to transmembrane insertion. Whereas this condition is already fulfilled at neutral and high pH, discharge of the histidine side chains is possible under acidic conditions only in the presence of other forces that favor membrane insertion (including hydrophobic energies). At acidic pH, the processes of membrane insertion (reorientation into the transmembrane configuration) and discharge are therefore tightly connected processes. When the X amino acids exhibit a low degree of hydrophobicity, the in-planar state remains populated even when the histidines are uncharged. In order to take into account the gradual protonation of four histidine side chains, the in-planar states IP1+,IP2+,IP3+, and IP4+ are taken into consideration (38Aisenbrey, C. (2003) Étude Topologique de Polypeptides Membranaires par RMN du Solide et Spectroscopie Infrarouge par Réflexion Totale Attenuée. Ph.D. thesis, University Louis Pasteur, Strasbourg, FranceGoogle Scholar). The equilibrium constant for the transition from IPo to TM is related to the Gibbs free energy ΔG gained by inserting the peptide into the membrane. The equilibrium constant is therefore defined as follows. kTM=[TM][IPo]=e-ΔGRT(Eq. 1) Furthermore, the uncharged in-planar configuration is in equilibrium exchange with the series of charged in-planar states, none of these being able to insert in a transmembrane fashion. When combined into a single state, [IP*] = [IP1+] + [IP2+] + [IP3+] + [IP4+], the constant (1/kCh) for the transition IPo ↔ IP* is the sum of the individual equilibrium constants. 1/kCh=[IP*][IPo]=∑1/kj(Eq. 2) By taking into consideration the number of possible states IPj+ and some algebraic transformation, the experimentally accessible ratio of the transmembrane over total peptide concentration, pTM, is given by the following (38Aisenbrey, C. (2003) Étude Topologique de Polypeptides Membranaires par RMN du Solide et Spectroscopie Infrarouge par Réflexion Totale Attenuée. Ph.D. thesis, University Louis Pasteur, Strasbourg, FranceGoogle Scholar), pTM=11+eΔGRT(1+e2.3c(pKa-pH))4(Eq. 3) with the meaning of c being discussed below. The resulting line shapes are shown in supplemental Fig. 8. The degree of hydrophobicity of the X amino acids and thus the ΔG of transfer from the in-plane to the transmembrane state is reflected by three properties of this function. First, the slope of the transition changes with ΔG, being steeper for high negative ΔG (very hydrophobic X). Second, the maximal amount of transmembrane orientation that is observed varies with ΔG. For moderately hydrophobic or hydrophilic peptides, the transmembrane state is never fully populated. Third, the transition midpoint is shifted toward pH values higher than the pKa values of the histidines. During the analysis of pH titration experiments, it should be considered that the local pH on the surface of a membrane can differ significantly from the pH in bulk solution. In addition, the protonation of the four histidines in the peptide are dependent on each other due to electrostatic interactions. We have, therefore, investigated the (de)protonation reactions of the LAH4X6 histidines in membrane environments by monitoring the pH-dependent 1H chemical shift changes of the histidines using solution NMR spectroscopy (see "Results" and Fig. 4). When analyzing the data, it becomes obvious that due to constraining the charges at biomolecular surfaces, the slope of the sigmoidal transition is reduced when compared with the Henderson-Hasselbach equation. The fitting procedure provides the averaged correction factor c, which is also used during the line-fitting analysis of the experimental transition curves using Equation 3. Whereas the fraction of transmembrane-oriented peptide can be evaluated by integration of the appropriate spectral ranges of the 15N solid-state NMR spectra, the ATR-FTIR data were analyzed using Equation 4 (31Bechinger B. Ruysschaert J.M. Goormaghtigh E. Biophys. J. 1999; 76: 552-563Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). 2pTM(RaATR-RbATR)+RbATR(RaATR+2)pTM(RbATR-RaATR)+RaATR+2(Eq. 4) Here RATRa and RATRb are the maximal and minimal dichroic ratio, respectively. The LAH4X6 peptides of the primary structure KKKKALXHLHXLAXHLHXLAXXALKKK-COOH were prepared by solid-phase peptide synthesis on a Millipore Corp. 9050 automatic peptide synthesizer using Fmoc chemistry (40Atherton E. Logan C.J. Sheppart R.C. J. Chem. Soc. Perkin Trans. I. 1981; : 538-546Crossref Google Scholar, 41Carpino L.A. Han G.Y. J. Org. Chem. 1972; 37: 3404Crossref Scopus (1091) Google Scholar). The six X-amino acids at positions 7, 11, 14, 18, 21, and 22 represent alanine (LAH4A6), glycine (LAH4G6), leucine (LAH4L6), tyrosine (LAH4Y6), or tryptophan (LAH4W6), respectively. The underlined alanine 13 position indicates the use of the 15N-labeled derivative of Fmoc-alanine (Promochem, Wesel, Germany) during the coupling step. Whenever the crude product was considered inadequate, the synthetic products were purified using reversed phase high performance liquid chromatography using an acetonitrile/water gradient and a Prontosil 300-5-C4 5.0-μm column (Bischoff Chromatography, Leonberg, Germany). The purity and composition of the peptides were controlled by reversed phase HPLC and MALDI mass spectroscopy. For solution NMR spectroscopy, 5 mg of peptide and 70 mg of perdeuterated DPC (Promochem, Wesel, Germany) were dissolved in 450 μl of 11 mm citrate-d5 (Campro, Emmerich, Germany) and 5 mm NaCl. The pH was adjusted using a 1 m NaOH stock solution. The NMR spectra were acquired on a Bruker AMX 500 spectrometer at 300 K. For the determination of the pH-dependent behavior of the histidine resonances, TOCSY (42Griesinger C. Otting G. Wüthrich K. Ernst R.R. J. Am. Chem. Soc. 1988; 110: 7870-7872Crossref Scopus (1193) Google Scholar) and NOESY spectra (43Macura S. Ernst R.R. Mol. Phys. 1980; 41: 95-117Crossref Scopus (1581) Google Scholar) were acquired using data matrices of size 2048 × 512 or 4096 × 512. Water suppression was achieved using the WATERGATE sequence (44Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3506) Google Scholar). Typically, the applied mixing times were 85 ms for NOESY and 60 ms for TOCSY spectra. Before Fourier transform, phase-shifted sine-square apodization functions and polynomial base-line corrections were applied. The processed matrix size was chosen between 2048 × 1024 and 4096 × 4096. Published methods were used to assign the resonances of the histidine side chains (45Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar). For solid-state NMR spectroscopy, 20 mg (∼6.5 μmol) of peptide was dissolved in water/trifluoroethanol and mixed with 300 mg (∼400 μmol) of POPC (Avanti Polar Lipids, Birmingham, AL). Prior to the addition of organic solvent, the pH of the peptide solution was adjusted by the addition of 1 n NaOH. The mixtures were slowly applied onto 30 thin cover glasses (11 × 22 mm), dried in air, and exposed to high vacuum overnight. After the samples had been equilibrated in an atmosphere of 93% relative humidity, the glass plates were stacked on top of each other and sealed. The uniaxially oriented stacks of membranes were introduced into the flat coil of a home-built solid-state NMR probe head (46Bechinger B. Opella S.J. J. Magn. Reson. 1991; 95: 585-588Google Scholar) with the bilayer normal parallel to the magnetic field direction. Proton-decoupled 15N solid-state NMR spectra were acquired on a wide bore Bruker AMX400 spectrometer using a cross-polarization pulse sequence (47Pines A. Gibby M.G. Waugh J.S. J. Chem. Phys. 1973; 59: 569-590Crossref Scopus (2344) Google Scholar). Typical acquisition parameters were as follows: spin lock time, 1.3 ms; recycle delay, 3 s; 1HB1-field, 1 millitesla; 254 data points; spectral width, 40 kHz. An exponential apodization function corresponding to a line broadening of 300 Hz was applied before Fourier transformation. The chemical shifts were referenced using 15N ammonium sulfate (27 ppm). ATR-FTIR spectroscopy of oriented membrane samples was performed using a Bruker IFS 55 infrared spectrometer equipped with a liquid nitrogen-cooled MCT detector, as described previously (31Bechinger B. Ruysschaert J.M. Goormaghtigh E. Biophys. J. 1999; 76: 552-563Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 38Aisenbrey, C. (2003) Étude Topologique de Polypeptides Membranaires par RMN du Solide et Spectroscopie Infrarouge par Réflexion Totale Attenuée. Ph.D. thesis, University Louis Pasteur, Strasbourg, FranceGoogle Scholar). In short, using trifluoroethanol/water solutions, 50 μg of peptide and 500 μg of POPC were spread onto an area of 5 × 1 cm of a carefully cleaned planar germanium plate (ACM, Villiers St. Frédéric, France). The crystal is characterized by an aperture angle of 45°, yielding 25 internal reflections. The organic solvent was evaporated under a stream of nitrogen. Oriented lipid bilayers spontaneously form along the surface of the crystal, as discussed in detail in Refs. 33Ivanov D. Dubreuil N. Raussens V. Ruysschaert J.M. Goormaghtigh E. Biophys. J. 2004; 87: 1307-1315Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar and 34Goormaghtigh E. Raussens V. Ruysschaert J.M. Biochim. Biophys. Acta. 1999; 1422: 105-185Crossref PubMed Scopus (508) Google Scholar. The pH was adjusted by covering the membranes with 200 μl of 66 mm phosphate buffer. After a few minutes, the buffer was carefully removed, and the sample was washed with 200 μl of MilliQ-water (Millipore). Although it remains possible that some of the peptide is washed away, thereby modifying the effective lipid-peptide ratio, this procedure has proven essential in order to obtain a well defined pH value without interference from the remaining salt. A fresh sample was prepared for every pH value. After the samples had been dried under a stream of nitrogen, the crystal was introduced into the ATR-FTIR spectrophotometer. During the measurements, the samples were kept dehydrated with a stream of nitrogen. During spectral acquisition, the spectrometer was continuously purged with dry air. For each spectrum, 64 scans were accumulated at a nominal spectral resolution of 2 cm–1. The peptides of the LAH4X6 series were designed to form α-helical secondary structures in membrane environments, as has been observed for many other sequences of related composition (27Bechinger B. 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This feature was confirmed by the large number of NH-NH nuclear Overhauser effect cross-peaks observed for the LAH4G6 peptides in the presence of detergent micelles (Fig. 2). Furthermore, when reconstituted into oriented POPC phospholipid bilayers, the LAH4X6 peptides exhibited absorptions at 1657 cm–1 (amide I) and at 1542 cm–1 (amide II) characteristic of helical structures (52Goormaghtigh E. Ruysschaert J.M. Molecular Description of Biological Membranes by Computer Aided Conformational Analysis.in: Brasseur R. CRC Press, Inc., Boca Raton, FL1990: 285-332Google Scholar) (Fig. 3). Some spectra of LAH4A6 showed small additional resonances at 1688 cm–1, suggestive of the presence of tiny amounts of β-sheet conformations.FIGURE 3The amide region of ATR-FTIR spectra of LAH4W6 reconstituted into oriented POPC membranes is shown for pH 6 (A) and pH 4 (B). The incident light was polarized in a parallel (dotted lines) or in a perpendicular orientation (dashed lines). The difference spectra (solid lines) are obtained by subtracting the perpendicular polarized spectrum from the parallel polarized spectrum after multiplication of the parallel polarized spectrum by 1.4. This factor corrects for the difference of strength of the evanescent power for the parallel and perpendicular polarizations as discussed in detail in Ref. 31Bechinger B. Ruysschaert J.