Specific interaction restrains structural transitions of an amphiphilic peptide in pulmonary surfactant model systems: An in situ PM-IRRAS investigation

Biochimica et Biophysica Acta 1798 (2010) 1263–1271

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a m e m

Specific interaction restrains structural transitions of an amphiphilic peptide in pulmonary surfactant model systems: An in situ PM-IRRAS investigation Hiromichi Nakahara, Sannamu Lee, Osamu Shibata ⁎ Department of Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan

a r t i c l e

i n f o

Article history: Received 18 January 2010 Received in revised form 6 February 2010 Accepted 12 February 2010 Available online 20 February 2010 Keywords: RDS Langmuir monolayer Lung surfactant DPPC DPPG SP-B

a b s t r a c t In situ polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) at the air–water interface has been used to determine secondary structure of the pulmonary surfactant model peptide, Hel 13-5, in the absence and the presence of phospholipid monolayers. Herein, fully saturated phospholipids of DPPC and DPPG are utilized to understand the effect of specific interaction between anionic DPPG and cationic Hel 13-5 on the peptide secondary structure. The spectrum frequency in the amide region (1500– 1700 cm− 1) obtained from PM-IRRAS has been confirmed by comparing with that from ATR-FTIR for the corresponding bulk films. The PM-IRRAS spectra of single Hel 13-5 monolayers indicate the α-helical contour in the amide region, which coincides with the result from CD measurements in aqueous solutions. In the presence of phospholipid monolayers, however, Hel 13-5 changes its conformation from the α-helix to the extended β-sheet as surface pressure increases upon compression at the interface, and this interconversion is found to be irreversible even during expansion process of monolayers. Furthermore, it is notable that the electrostatic interaction between DPPG and Hel 13-5 inhibits to some extent the interconversion to the β-sheet during compression. These features are completely different from the bulk behavior, which demonstrates different roles of native proteins in the bulk phase and at the interface for pulmonary functions. In addition, the conformational variation of Hel 13-5 does not indicate close correlation with surface activity, which is common characteristic even for reversible hysteresis curves in pulmonary surfactant systems. This suggests that the secondary structure of native proteins is not strongly related to the surface activity during respiration. This work contributes to secondary structure determination of Hel 13-5 in the phospholipid domains in situ at the air–water interface and will provide insight into the molecular and physiological mechanism for SP-B and SP-C actions across the interface. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Pulmonary surfactant (PS) is a surface-active material coating the air-alveolar fluid interface of mammalian lungs. It plays an important role in vital physiological process such as respiratory movement. Its main function is in vivo to lower surface tension at the surface, preventing alveolar collapses at end-expired state and minimizing the work of breathing [1]. This function is achieved by formation of lipid monolayers highly enriched in dipalmitoylphosphatidylcholine (DPPC) and bilayer or multilayer structures (surface-associated reservoirs) closely attached to the monolayers. The existence of the layered structures has been reported in vitro by fluorescence microscopy (FM) and atomic force microscopy (AFM) [2–5] and also in vivo by electron microscopy [6]. PS deficiency suffers neonatal respiratory distress syndrome (NRDS) in premature infants, whereas pathological alternation suffers acute respiratory distress syndrome ⁎ Corresponding author. Tel./fax: +81 956 20 5686. E-mail address: [email protected] (O. Shibata). URL: http://www.niu.ac.jp/~pharm1/lab/physchem/indexenglish.html (O. Shibata). 0005-2736/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2010.02.018

(ARDS) in adults. Exogenous surfactant preparations extracted from bovine or porcine lungs are commonly administered to NRDS patients for their therapy. Although the preparations above are dramatically effective for the patients, they are involved in the risk of animal infections such as bovine spongiform encephalopathy (BSE), potential viral contamination, and inherent immunity. Other drawbacks include a costly purification procedure and the difficulty of producing batchto-batch uniformity. Therefore, the preparations fully made of synthetic surfactants with proteins (or peptides) have been desired in the clinical surfactant replacement therapy for NRDS and ARDS. PS is composed of lipids (∼90 wt.%) and proteins (∼ 10 wt.%). Herein the lipids consist mainly of phosphatidylcholines (especially DPPC, ∼50 wt.%) and of smaller but significant amounts of phosphatidylglycerol (PG) [7–9]. With respect to the proteins, the hydrophobic surfactant protein B (SP-B) has a crucial role in pulmonary functions [10–12]. The human SP-B has a net charge of +7, which is thought to be essential for electrostatic interactions between its polar residues and the head groups of negatively charged phospholipid such as PG [4,13]. Due to the importance of SP-B for proper PS activity, synthetic peptides based on amino acid sequence of SP-B have been designed to

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clarify the mechanism of lipid–protein interactions around the interface and to develop synthetic surfactant preparations containing artificial peptides such as KL4 [14–16] and Hel 13-5 [17–19] for the RDS patients. Hel 13-5 is a monomeric synthetic peptide based on N-terminal segment of human SP-B. Its structure and surface activity have systematically been investigated employing the Langmuir monolayer technique in combination with a wide variety of morphological observations. The secondary structure of Hel 13-5 is predominantly α-helix in the aqueous solutions from the CD measurements [20,21]. In addition, according to the extrapolated molecular area of surface pressure (π)–area (A) isotherm for Hel 13-5 monolayers, it is also considered to adopt α-helical structures at the interface [22]. However, the direct proof of the secondary structure of Hel 13-5 in situ at the interface has not been performed yet using the spectroscopic technique such as infrared external reflectance spectroscopy. To acquire molecular information on protein or peptide secondary structure directly from the lipid and protein (peptide) constituents of monolayers at the air–water interface, the use of infrared reflection absorption spectroscopy (IRRAS) was originally developed in the mid1980s, and the progress in this field has been reviewed [23,24]. Then the IRRAS technique has been applied to the study on monolayers of extracted pulmonary surfactant preparations [25]. In addition, it has been shown that the amide vibrations of proteins can be observed in pure or highly enriched lipid–protein monolayers to obtain orientational and conformational information on surfactant proteins upon compression. Although these studies have contributed significantly to clarification of protein or peptide secondary structures and orientations, some difficulties for the Langmuir film technique still remain. In particular, the band intensity is inherently low in IRRAS at the air–water interface, and the strong absorption of the water vapor hides the spectral region in which the most interesting molecular information is included. Even though the interferometer becomes now very stable and the detector becomes sensitive, the conventional IRRAS requires long acquisition times and accurate control of vapor pressure during sample and reference spectrum measurements. From the mid-1990s, in order to overcome these problems, a differential IR reflectivity technique by the manner that the polarization of the incident electric field is rapidly modulated between the p- and s-channels (PM-IRRAS) was developed [26–28]. This technique is almost insensitive to the strong IR absorption of water vapor and allows the extraction of faithful information on conformation and orientation of protein in the monolayer. It is evident that secondary structure determination of surfactant proteins and their analogue peptides in monolayers is essential for understanding their in vivo function during respiration and designing model peptides for the replacement therapy. Recently, the determination of secondary structures at the interface has been performed to clarify the mechanism of the secondary structure conversion of the prion and the amyloid proteins from the normal into the abnormal form, which is the main cause of several human and animal diseases such as Creutzfeldt-Jakob and Alzheimer's disease [29–32]. It is noticed that the peptide secondary structure determined in the bulk phase differs from that found in the monolayer state [33,34]. Therefore, proving the secondary structure of protein or peptide monolayers in the lipid environment in situ at the air– water interface is quite important and essential. Nevertheless, only a few studies employing the PM-IRRAS technique have been attempted directly to determine the conformation of native pulmonary surfactant proteins and the mimicking peptides in the monolayer state at the interface [35–37]. In the current work, we report that Hel 13-5 in the phospholipid environment exhibits an irreversible conformational change from the α-helix to the antiparallel β-sheet using the in situ PM-IRRAS technique at the air–water interface. The obtained spectra from PM-IRRAS are analyzed and assessed by comparing with the spectra from attenuated total reflectance (ATR)-FTIR for the corresponding dry films. The aim of this study is to elucidate the secondary structure variation of Hel 13-5 against surface pressure, physical process of lateral compression and

successive expansion, and the electrostatic interaction with anionic phospholipids [19]. We present multiple IR spectroscopic data for pulmonary surfactant model preparations of DPPC, dipalmitoylphosphatidylglycerol (DPPG), and DPPC/DPPG (= 4/1, mol/mol) with or without Hel 13-5 under the physiological condition of pH and ionic strength [19]. This work indicates that Hel 13-5 secondary structure in phospholipid monolayers strongly depends upon phospholipid charge and surface pressure, which is not consistent with the results of the previous CD experiment in the bulk phase [20,21]. 2. Materials and methods 2.1. Materials The Hel 13-5 (NH2-KLLKLLLKLWLKLLKLLL-COOH, see Fig. S1 in the Supporting Material) peptide was synthesized by the Fmoc (9-fluorenylmethoxycarbonyl) technique and purified with reversephase HPLC as described elsewhere [20]. More detailed procedures of synthesis, purification, and basic analysis for Hel 13-5 were reported previously [21,38]. L-α-Dipalmitoylphosphatidylcholine (DPPC, purity N99%) and L-α-dipalmitoylphosphatidylglycerol (DPPG, purity N99%) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). DPPG was supplied as its sodium salt. These lipids were used without further purification or characterization. Chloroform (99.7%) and methanol (99.8%) used as spreading solvents were purchased from Cica-Merck (Uvasol, Tokyo, Japan) and nacalai tesque (Kyoto, Japan), respectively. Tris(hydroxymethyl) aminomethane (Tris) and acetic acid (HAc) of guaranteed reagent grade for the preparation of a subphase were obtained from nacalai tesque. Sodium chloride (nacalai tesque) was roasted at 1023 K for 24 h to remove all surface-active organic impurities. The substrate solution was prepared using thrice distilled water (the surface tension = 71.96 mN m− 1 at 298.2 K and the electrical resistivity = 18 MΩ cm). 2.2. Langmuir monolayer preparations Stock solutions of DPPC (1.0 mM), DPPG (0.5 mM), and Hel 13-5 (0.1 mM) were prepared in chloroform/methanol (2/1, v/v), and the binary and ternary solutions are prepared by mixing these mother solutions. In the present work, we used three model lipids; DPPC, DPPG, and the binary DPPC/DPPG mixture with a fixed ratio (4/1, mol/mol). For the pulmonary surfactant model preparations, the lipids with a constant and critical amount of Hel 13-5 (XHel 13-5 = 0.1) were utilized because peptide concentrations strongly affect the phase behavior [19] and spectrum mode [33] of pulmonary surfactant preparations. More detailed information on lipid and peptide compositions was described in the previous study [19]. An aliquot of the solutions is spread onto the subphase of 0.02 M Tris buffer with 0.13 M NaCl (pH 7.4). The preparation of the subphase was performed as described previously [17,22]. The spreading solvents were allowed to evaporate for 30 min prior to compression for PM-IRRAS measurements. The monolayer was compressed and expanded at a speed of b0.11 nm2 molecule− 1 min− 1. The temperature was kept constant at 298.2 K within ± 0.1 K throughout the experiments. 2.3. ATR-FTIR measurements Polarized ATR-FTIR spectra were recorded on a Fourier transform spectrometer (JASCO FT/IR-4200, Tokyo, Japan) at a nominal resolution of 2 cm− 1. A horizontal ATR accessory (ATR PRO410-S, JASCO) was used with a germanium (Ge) crystal as an internal reflection element (IRE). The angle of incidence of the IRE was 45° (single reflection). The surface of the ATR crystal available for coating by the sample had dimensions of 1.8 mm2. A zinc selenide (ZnSe)-mounted, IR holographic wire-grid polarizer with the diameter of 25 mm (Edmund Optics, NJ, USA) was used in the

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incident beam to control both p- (parallel to the plane of incidence) and s- (perpendicular) polarization. A total of 512 individual scans were co-added at room temperature. The spectrometer was purged with dry air (1.0 l/min). Thirty-microliter aliquots of ∼ 0.5 mg/ml CHCl3/MeOH (= 2/1, v/v) mixed solutions of the desired sample composition were dried down on the Ge ATR crystal. After evaporation of the solvents, spectra of the dry sample (not hydrated vesicles) were recorded with the polarized (p and s) and nonpolarized radiation. Data analyses of the phosphate stretching (1000–1300 cm− 1), the amide (1500–1700 cm− 1), and the methylene stretching (2800–3000 cm− 1) regions were accomplished with Spectra Manager Ver. 2 (JASCO). The spectra displayed in the figures were the representatives of 3 runs at least.

1662–1645 cm− 1 [42]. The dichroic ratio is defined as the ratio between the absorption of light polarized parallel and the one perpendicular to the surface of the IRE. For the α-helix of Hel 13-5, the RATR was determined using the integrated peak areas of the α-helical marker band in the amide I region. Before the area calculations, a straight baseline passing through the ordinates at 1700 and 1600 cm− 1 was subtracted. This method of correction for the contribution of the baseline to the integrated peak areas for amide I seems reasonable since no significant peptide absorption occurs at these designated wavelengths. This ratio was then used to calculate the molecular order parameter (S) of the peptide as follows [43]:

S=

2.4. PM-IRRAS measurements In situ polarization modulation infrared reflection absorption spectrum (PM-IRRAS) measurements at the air–water interface were performed using a KSV PMI 550 instrument (KSV Instruments Ltd., Helsinki, Finland) coupled to a commercially available film balance system (KSV Minitrough, KSV Instruments Ltd.). The PMI 550 instrument contains a Fourier transform IR spectrometer equipped with a PM unit on one arm of a goniometer and a HgCdTe (MCT) detector on the other arm. The IR beam was conducted out of the spectrometer and was directly steered into the interface by using no mirrors or external reflection accessory. The incident angle at 80° relative to the normal of the air–water interface was selected in the present study because of the highest signal-to-noise (S/N) ratio in between the incident angles of 75° and 80° [39,40]. The incident IR beam was modulated by a ZnSe photoelastic modulator (PEM-100, Hinds Instruments, Hillsboro, OR) operating at its resonance frequency of 50 kHz, with the frequency at 1500 cm− 1 and a retardation of λ/2. An operation of the IR spectrometer and the PEM allows separation of two signals at the MCT detector using dual-channel electronics with lock-in detection, the sum (or reference spectrum) and difference components (or surface specific information), respectively. The simultaneous measurement of both spectra results in a prominent reduction of the effect of water vapor on the spectrum regions of the amide I and II. The bare water surface of 0.02 M Tris buffer with 0.13 M NaCl (pH 7.4) was used as the background, and the film-covered surface was used as the sample. The PM-IRRAS signal (S) was obtained from the following equation: S = ΔR/R = (Rp − Rs)/(Rp + Rs), where Rp and Rs are the parallel (p)- and perpendicular (s)-polarized reflectances to the plane of incidence, respectively [41]. The normalized PM-IRRAS spectrum is displayed by ΔS = (S − S0) / S0, where S0 is the signal of the bare water surface and S is that of the film-covered surface. The total acquisition time for each spectrum was 5 min, resulting in 3000 interferograms per spectrum. The spectral range of the PMI 550 device is 800–4000 cm− 1, and the resolution is 8 cm− 1. The spectra displayed in the figures were the representatives of 5 runs at least. The raw normalized spectra before subtracting the fitting base line and peak processing are displayed in the figures. Spectra were subjected to second-derivative and curve-fitting procedures in order to fit the amide I and II bands in the region 1500– 1700 cm− 1 with a Gaussian band shape, using PeakFit software (ver. 4.12, SeaSolve Software Inc., CA, USA). Fitting was judged acceptable when the value of coefficient for the determination between the simulated and the experimental (original) spectra was more than 0.99. The nonlinear curve fitting analysis provided information on peak intensity, peak position, full width at half height (FWHH), and integrated peak area for the Gaussian components in the amide region. 2.5. Secondary structure orientation from polarized ATR-FTIR spectra The order parameter of an α-helical peptide in a supported film can be derived from the measured dichroic ratio (RATR) in the spectral region

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Ex2 −RATR Ey2 + Ez2 2 ⋅ 3 cos2 α−1 Ex2 −RATR Ey2 −2Ez2

ð1Þ

where Ex, Ey, and Ez denote the normalized electric field components when the IR beam is polarized in the respective dimensions. For the peptide, the angle (α) between the helix axis and the transition dipole moment, 38–40°, was adopted in the present study [43–47]. For the phospholipids, the angle (α′) between the acyl chain axis and the transition dipole moment, 90°, was adopted in the present study [46,48]. The values of Ex, Ey, and Ez depend on whether the thickness of the deposited film is greater or smaller as compared with the penetration length of the evanescent wave in the ATR-FTIR experiment. The decay depth of the evanescent wave is typically of the order of 0.5 μm. At the wavelength of the amide I absorption, the penetration depth is 0.4 μm, which extends far beyond the thickness of our thin film (b ∼0.1 μm). Thus, Ex = 1.411, Ey = 1.461, and Ez = 0.770 for the sample configuration are used in the present study [49]: angle of incidence of IR (45°), refractive indices of the IRE (n = 4.0 for Ge), refractive index of the air (n = 1.0), refractive index of the sample (n = 1.4 [43,50]). In addition, S is also calculated using the following equation;

S=

3 cos2 θ−1 2

ð2Þ

where θ is the mean angle between the helix axis and the membrane normal, namely the mean tilt angle of α-helix backbone against the direction perpendicular to the IRE surface. Combination of Eqs. (1) and (2) could provide the θ values in the absence and presence of the phospholipids. The maximum standard deviation of θ for the α-helical marker band in the amide I region was 7° in the present manner. In fact, this manner was performed under many assumptions [51]. Therefore, the resultant angles were utilized as the complementary information in the present study. 3. Results 3.1. Polarized ATR-FTIR spectra Polarized ATR-FTIR spectra of Hel 13-5 films in the 1400–1800 cm− 1 region including the amide I (1600–1700 cm− 1) and II (1500– 1600 cm− 1) regions are shown in Fig. 1. The intense bands in this region are the two positive bands at 1656 (amide I) and 1541 (amide II) cm− 1. It is well established that the frequency and shape of the amide band depend critically on the secondary structure of peptide and protein films [44,48,50]. Both of the peaks observed are indicatives of the α-helical structure. This shows that the secondary structure of pure Hel 13-5 in the dry-film form is completely α-helix. The p-polarized spectrum is wholly larger in peak intensity than the s-polarized one, which reflects the difference in absorbance of the two polarized incident radiation due to the tilted or oriented molecular backbone to the Ge surface. The difference is commonly related to a dichroic ratio to calculate the molecular orientation.

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Fig. 2 shows polarized ATR-FTIR spectra of dry films of the DPPC/Hel 13-5, DPPG/Hel 13-5, and DPPC/DPPG (= 4/1, mol/mol)/Hel 13-5 systems at the fixed molar ratio (XHel 13-5 = 0.1) in the 1400– 1800 cm− 1 region. In the DPPC/Hel 13-5 spectra, four major peaks are observed at ∼ 1745, 1659, 1543, and 1467 cm− 1. The peaks at ∼ 1745 and 1467 cm− 1 are assigned to the C = O stretching and CH2 bending vibrations of DPPC, respectively. Both the amide bands at ∼ 1659 and 1543 cm− 1 are indicatives of the α-helix. However, the amide I feature is accompanied by a shoulder appearing around 1680 cm− 1 as shown by arrows. This suggests that Hel 13-5 also takes on an antiparallel β-sheet [52–54]. Considering no shoulder like that in the single Hel 13-5 spectra, the β-sheet formation is found to be induced by environmental DPPC molecules. As for the DPPG/

Hel 13-5 and DPPC/DPPG/Hel 13-5 systems, the spectra are quite similar in peak position to those of the DPPC/Hel 13-5 system. The ATR-FTIR spectra in the amide I and II regions indicate that the secondary structure of Hel 13-5 in the phospholipid components is predominantly the α-helix. However, Hel 13-5 partly transforms from the α-helix to the β-sheet structure due to the interaction with the phospholipids. To elucidate the electrostatic interaction between charged moieties of the phospholipids and Hel 13-5 [19], attention is focused on the spectra in the region of phosphate stretching bands of phospholipids (1000–1300 cm− 1). Fig. 3 shows the non-polarized ATR-FTIR spectra of dry films of DPPC, DPPG, and DPPC/DPPG (=4/1, mol/mol) with (normal line) or without Hel 13-5 (dot line). There are several bands in the DPPC/Hel 13-5 spectra. Namely, the bands at ∼1242, 1200, 1175, 1094, and 1071 cm− 1 are assigned to the asymmetric PO2 stretching [23,49], C–N stretching [49], asymmetric CO–O–C stretching [55], symmetric PO2 stretching [23,49], and symmetric CO–O–C stretching vibrations [55], respectively. Although the peak intensity varies in between the spectra of pure DPPC and DPPC/Hel 13-5 due to the difference of sample amounts on the Ge surface, the peak positions assigned above remain almost the same. In the case of the DPPG/Hel 13-5 system, a different trend from the DPPC/Hel 13-5 system is observed. Besides the symmetric PO2 stretching mode at ∼ 1090 cm− 1, the spectrum of pure DPPG films has a peak at ∼ 1054 cm− 1, which is assigned to charged C–O–P stretching vibration [55,56]. However, the band shifts from 1054 to 1077 cm− 1 by the addition of Hel 13-5 to DPPG. In common, it is well known that if the charged phosphate group is protonated, the absorption peak of the stretching C–O–P or PO2 modes shifts to lower wave number (1010–1040 cm− 1) [55]. In contrast, the band of the stretching C–O–P mode shifts to larger wave number in the present system. Considering that the peak shift of the certain vibration in the spectroscopy means a change in orientation and conformation of the corresponding functional group, the shift observed in the present study may suggest the specific interaction between the negatively charged PG head group of DPPG and the positively charged moiety of Hel 13-5. As for the DPPC/DPPG/Hel 13-5 system, the spectra are quite similar in peak

Fig. 2. Polarized ATR-FTIR spectra of a dry film of DPPC/Hel 13-5 (upper), DPPG/Hel 13-5 (middle), and DPPC/DPPG (=4/1, mol/mol, lower)/Hel 13-5 systems at XHel 13-5 = 0.1 in the region of 1400–1800 cm− 1 at room temperature. Solid lines correspond to p-polarization, and dashed lines, to s-polarization of the incident radiation.

Fig. 3. Non-polarized ATR-FTIR spectra of a dry film of DPPC/Hel 13-5 (upper), DPPG/ Hel 13-5 (middle), and DPPC/DPPG (= 4/1, mol/mol, lower)/Hel 13-5 systems in the region of 1000–1300 cm− 1 at room temperature. Solid lines correspond to the spectra in the presence of Hel 13-5 (XHel 13-5 = 0.1), and dotted lines, to the spectra in the absence of Hel 13-5 (XHel 13-5 = 0).

Fig. 1. Polarized ATR-FTIR spectra of a dry film of Hel 13-5 in the region of 1400– 1800 cm−1 at room temperature. Solid line corresponds to p-polarization, and dashed line, to s-polarization of the incident radiation.

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position to those of the DPPC/Hel 13-5 system. That is, the peak shift seen in the DPPG/Hel 13-5 system is not observed due to the presence of small amount of DPPG in the ternary mixture. Focusing on orientations of the Hel 13-5 α-helix backbone, a dependency of the angle (α) between the helix axis and the transition dipole moment on the mean tilt angle (θ) of Hel 13-5 backbone is shown in Table S1 in the Supporting Material. Although the θ values slightly vary with the angle (α), the changes are not significant to elucidate the orientation. The mean tilt angle for pure Hel 13-5 films is found to be ∼ 59° from calculation with the dichroic ratio. This value indicates that Hel 13-5 backbone oriented closer to the plane of the Ge surface. The angles in the presence of DPPC (57–58°) and the DPPC/ DPPG mixture (∼ 58°) are similar to that of pure Hel 13-5. However, the DPPG/Hel 13-5 system generates a significantly different value of 70–72° from the other systems, indicating the orientation almost parallel to the Ge surface. As for the phospholipids, the mean tilt angles of their acyl chains are ∼49° for DPPC/Hel 13-5, ∼50° for DPPG/Hel 13-5, and ∼34° for DPPC/DPPG/Hel 13-5, which indicates that Hel 13-5 backbones incline more than phospholipid acyl chains. The above difference in tilt angle is due to the difference in electrostatic interaction between DPPG and Hel 13-5. Just for reference, the mean tilt angle calculated from the PM-IRRAS spectra is shown in Table S2 in the Supporting Material. Although the values are not somewhat reliable due to the lower accuracy and intensity of PM-IRRAS peaks compared with ATR ones, they provide supportable information for the above results. The angles in Table S2 imply the orientation parallel to the air–water interface for Hel 13-5 monolayers. 3.2. In situ PM-IRRAS spectra of Hel 13-5 monolayers Shown in Fig. 4 are the PM-IRRAS spectra (1400–1800 cm− 1) of Hel 13-5 monolayers on 0.02 M Tris buffer with 0.13 M NaCl (pH 7.4) at 298.2 K. These PM-IRRAS spectra were acquired while the monolayer was held at selected surface pressures from 5 to 45 mN m− 1 (for Hel 13-5 alone) during step-wise compression. Pure Hel 13-5 forms a disordered (expanded) monolayer on the buffer subphase, and its monolayer collapse pressure is ∼42 mN m− 1 (see Fig. S2 in the Supporting Material) [5,22]. At 5 mN m− 1, the predominant intensity

Fig. 4. In situ PM-IRRAS spectra of monolayers of Hel 13-5 alone as a function of surface pressure (π) in the region of 1400–1800 cm− 1 on 0.02 M Tris buffer with 0.13 M NaCl (pH 7.4) at 298.2 K. The spectrum at 45 mN m− 1 reflects films above the collapse pressure of Hel 13-5 (πc = ∼42 mN m− 1 [5,22]).

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of the broad amide I band is seen at ∼1660 cm− 1, corresponding to the α-helix secondary structure. In addition, the indicative of the α-helix is also observed in the amide II region at ∼ 1539 cm− 1. The surface selection rules of the PM-IRRAS experiment at the air–water interface specify that strong positive absorption bands in the spectrum indicate IR transition dipole moments, which are aligned parallel to the interface [41]. Therefore, the α-helical backbone of Hel 13-5 is found to be oriented parallel to the surface. With increasing surface pressure to 40 mN m− 1, the peak intensity of the amide I and II regions slightly increases due to the enrichment of surface concentration of Hel 13-5 upon lateral compression. Above the collapse pressure of Hel 13-5 monolayers, the spectrum in the amide I region at 45 mN m− 1 clearly indicates two different peaks at ∼1645 and ∼1612 cm− 1. The former band is apparently seemed to be characteristic of the α-helix. However, the peak frequency somewhat shifts to lower wave number. This small shift means that the α-helical structure of Hel 13-5 is exposed by the surrounding water molecule [52,57]. The latter band is assigned to an extended forms including the antiparallel β-sheet [33,54]. Overcompression of Hel 13-5 monolayers induces the structural interconversion from the α-helix to β-sheet. 3.3. In situ PM-IRRAS spectra of phospholipids–Hel 13-5 systems PM-IRRAS spectra in the region of 1400–1800 cm− 1 acquired from DPPC monolayers containing Hel 13-5 (XHel 13-5 = 0.1) on 0.02 M Tris buffer with 0.13 M NaCl (pH 7.4) at several surface pressures (π) during compression and expansion are shown in Fig. 5 (see Fig. S3 in the Supporting Material). In comparison to Fig. 4, the peak intensities in the amide I and II regions become less due to the small surface concentration of Hel 13-5. To overcome the defect, peak separation to the Gaussian components is faithfully accomplished according to the manner mentioned in Materials and methods. At 5 mN m− 1, the intense constituents of the amide I (at ∼1653 cm− 1) and II (at ∼1534 cm− 1) modes are assigned to the α-helix. A similar spectral feature is also observed at 25 mN m− 1. As surface pressure increases toward 60 mN m− 1, the peak assigned to the α-helix almost disappears. Instead, the component near ∼ 1617 cm− 1, which is characteristic of the antiparallel β-sheet, appears in the spectra, and

Fig. 5. In situ PM-IRRAS spectra of monolayers of the DPPC/Hel 13-5 system (XHel 13-5 = 0.1) as a function of surface pressure (π) during compression and successive expansion (exp.) in the region of 1400–1800 cm− 1 on 0.02 M Tris buffer with 0.13 M NaCl (pH 7.4) at 298.2 K. The marker band regions of the α-helix and the β-sheet in the amide I contour are emphasized in the dashed rectangles.

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the peak intensity of the β-sheet marker band relative to the α-helical marker band increases. During expansion process of the monolayers from 60 to 25 mN m− 1, the β-sheet marker band decreases in peak intensity and the peak assigned to the hydrated α-helix at ∼ 1642 cm− 1 emerges in the spectra. However, the intensity ratio of the α-helix marker band relative to the β-sheet one does not come back to the original ratio during compression. It suggests that the interconversion of the α-helix into the β-sheet is not completely reversible. To understand the interconversion of Hel 13-5 secondary structures in the phospholipid environments in terms of the electrostatic interaction, we additionally examined Hel 13-5 in DPPG and DPPC/DPPG (=4/1, mol/mol) monolayers at XHel 13-5 = 0.1 (see Fig. S3). PM-IRRAS spectra of the 1400–1800 cm− 1 region for the monolayer of DPPG/Hel 13-5 and DPPC/DPPG/Hel 13-5 during compression and expansion are shown in Figs. 6 and 7, respectively. Under the compression process, the β-sheet marker bands near ∼ 1606 cm− 1 gradually emerge in the spectra with increasing surface pressure for the both monolayers, similarly to the DPPC/Hel 13-5 preparation (Fig. 5). However, the minor α-helix marker bands remain in the spectra of the DPPG/Hel 13-5 and DPPC/DPPG/Hel 13-5 systems even at higher surface pressures. This may imply that the specific interaction between DPPG and Hel 13-5 disturbs the interconversion of the Hel 13-5 secondary structure into the β-sheet upon compression. During the subsequent expansion of the monolayers, the β-sheet marker bands near 1610 cm− 1 are still left in the spectra for the both systems (Figs. 6 and 7). It is interesting to note that the interconversion of Hel 13-5 secondary structure from the α-helix into the β-sheet is an irreversible process even under a phospholipid environment. As for the lower frequency region for the lipid phosphate group, the peak shift takes place in the presence and absence of Hel 13-5 for only the DPPG/Hel 13-5 system (data not shown), similarly to the ATR-FTIR result. The addition of Hel 13-5 to DPPG monolayers induces the band shift from ∼1055 (charged C–O–P) to 1060 cm− 1. The peak shift by ∼ 5 cm− 1 from PM-IRRAS is smaller than that from ATR-FTIR (∼ 23 cm− 1). This resulted from the

Fig. 6. In situ PM-IRRAS spectra of monolayers of the DPPG/Hel 13-5 system (XHel 13-5 = 0.1) as a function of surface pressure (π) during compression and successive expansion (exp.) in the region of 1400–1800 cm− 1 on 0.02 M Tris buffer with 0.13 M NaCl (pH 7.4) at 298.2 K. The marker band regions of the α-helix and the β-sheet in the amide I contour are emphasized in the dashed rectangles.

Fig. 7. In situ PM-IRRAS spectra of monolayers of the DPPC/DPPG (= 4/1, mol/mol)/ Hel 13-5 system (XHel 13-5 = 0.1) as a function of surface pressure (π) during compression and successive expansion (exp.) in the region of 1400–1800 cm− 1 on 0.02 M Tris buffer with 0.13 M NaCl (pH 7.4) at 298.2 K. The marker band regions of the αhelix and the β-sheet in the amide I contour are emphasized in the dashed rectangles.

formation of hydrogen band networks between DPPG head groups and water molecules in the subphase as opposed to the dry-film condition. 3.4. Analysis of surface pressure dependence of secondary structure transition of Hel 13-5 The α-helix ratio of Hel 13-5 relative to the β-sheet component against surface pressure for the DPPC/Hel 13-5, DPPG/Hel 13-5, and DPPC/DPPG (=4/1, mol/mol)/Hel 13-5 monolayers at XHel 13-5 = 0.1 is clearly displayed in Fig. 8. The ratio at different π values was calculated from integrated peak areas of the α-helix (∼ 1650 cm− 1) and of the β-sheet (∼1620 cm− 1) marker bands in the amide I region; the term α/(α + β) means the peak area ratio of amide I band intensities for ∼1650/(∼ 1650 + ∼ 1620) cm− 1 [33]. In the DPPC/Hel 13-5 system (Fig. 8A), the percentage of the α-helical component decreases from 50–80% down to near 0% with increasing surface pressure during compression of the monolayers. Then, upon subsequent expansion, the percentage immediately recovers up to ∼30% and the value is kept almost constant as surface pressure reduces. This quantitatively confirms the irreversible interconversion of Hel 13-5 secondary structures from the α-helix into the β-sheet. As for the DPPG/Hel 13-5 monolayer (Fig. 8B), it is quite interesting to note that the percentage of the α-helix marker band reduces from ∼ 60% down to ∼30%, not to ∼ 0% upon compression. Namely, the interconversion into the β-sheet is restrained by the surrounding DPPG monolayer, which may result from the electrostatic interaction between both components. During the subsequent expansion, the percentage does not come back to the initial value but maintains the same value (∼30%) up to lower surface pressures as is the case for the DPPC/Hel 13-5 monolayer. As for DPPC/DPPG/Hel 13-5 (Fig. 8C), which has physiologically more relevant phospholipid compositions, it is notable that the variation in percentage against surface pressure during compression and expansion resembles that for the DPPG/Hel 13-5, not that for the DPPC/Hel 13-5 system. Considering that DPPG is much smaller in amount in the ternary system than DPPC, the presence of a small amount of DPPG plays a critical role in the inhibition of the interconversion of Hel 13-5 secondary structure.

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Fig. 8. Peak area ratios [α/(α + β)] of the amide I band intensity for ∼1650/(∼1650 + ∼1620) cm− 1 as a function of surface pressure (π) during compression (solid circles and solid lines) and expansion (open cycles and dashed lines), as obtained from curve fitting to PM-IRRAS spectra. ∼1650 cm− 1 is assigned to the α-helical secondary structure; ∼ 1620 cm− 1 is assigned to the antiparallel β-sheet. (A) DPPC/Hel 13-5. (B) DPPG/Hel 13-5. (C) DPPC/DPPG (= 4:1, mol/mol)/Hel 13-5 at XHel 13-5 = 0.1.

4. Discussion PM-IRRAS has several advantages more than conventional polarized IRRAS for the study on monolayers at the air–water interface. In particular, PM-IRRAS has the ability to discriminate isotropic water molecules and ones adsorbed from vapor and to analyze the resulting spectra directly for the orientation and conformation of the surface monolayer [26]. However, there are a few considerable disadvantages in the PM-IRRAS measurement applied to monolayers. One of the disadvantages is that the absence of an absorbance signal does not necessarily indicate the absence of monolayers at the interface. In contrast, the signal absence might also indicate an isotropic monolayers or a preferred monolayer orientation in which the transition dipole moment is oriented such that co-existing positive and negative signal contributions cancel one another [26]. Therefore, the combination with other spectroscopic techniques such as transmission IR and ATR IR is indispensable to accomplish correct interpretation and analysis of the resulting PM-IRRAS spectra. In the present study, we combine PM-IRRAS and polarized ATR-FTIR measurements in order to elucidate the Hel 13-5 secondary structure in situ at the air–water interface. Although the peak intensity in the amide region of PM-IRRAS spectra is relatively low due to the small surface concentration of Hel 13-5, the corresponding absorbance frequency nearly coincides with that measured by the ATR-FTIR technique. This result strongly

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compensates for the defects of the PM-IRRAS technique, and the spectra in the present work could correctly reflect in situ molecular vibration modes. Moreover, in the present study, only the Langmuir monolayer is treated to overcome the present objective. In common, the spreading monolayer shows a different property from the adsorbed monolayer, which more faithfully mimics the pulmonary membrane in the alveoli. However, application of the in situ PM-IRRAS method to the adsorbed monolayer makes it difficult and complicated to understand molecular mechanism in pulmonary functions; unexpected peak information besides the desired monolayers' vibration is likely to be accumulated due to the existence of molecules near the interface and a quantitative analysis is difficult to be performed due to the unclearness of surface concentration of the sample. Thus, the Langmuir monolayer was herein utilized as a model of monolayers at the air-alveolar fluid interface. Pulmonary surfactants exert a crucial function related not only to respiratory movement but also to its work reduction at the air-alveolar interface. In the physiological process, the surfactants adsorb to the interface from the alveolar hypophase through conformational transformations such as lamellar body, tubular myelin, and other aggregations, forming an interfacial monolayer. In contrast, synthetic PS preparations adsorb to the interface from the alveolar-air space in the case of PS replacement therapy. Therefore, it is very important to determine the secondary structures of Hel 13-5, which is the surface lining model peptide of SP-B, in the monolayer state in situ at the air– water interface. The present work is surely aimed to understand the molecular mechanism of Hel 13-5 surface mode and to design improved versions of the mimicking peptide. Lee and co-workers have reported that the secondary structure of Hel 13-5 in the aqueous solutions in the presence of various phospholipids is the α-helical form of ∼ 75–100% by CD measurement [20,21]. So far, however, it has not been clarified yet what type of the secondary structure does Hel 13-5 form at the interface. The current PM-IRRAS spectra provide a direct evidence of Hel 13-5 secondary structure in the monolayer at the surface. The Hel 13-5 secondary structures have a direct influence on charge of phospholipid head groups and surface pressure. In all the mixtures containing the phospholipids, the lateral compression of the monolayer induces the interconversion of Hel 13-5 secondary structure from the α-helix to the antiparallel β-sheet structure. Considering that the predominant β-sheet signal is not adopted in the CD spectra for the Hel 13-5 aqueous solution, driving forces of the structure conversion to the β-sheet are thought to be a heterogeneous intermolecular interaction and an energy gain induced by lateral compression [52]. As for native SP-B and SP-C, it has been reported that the two proteins adopt a variety of conformations including α-helix and β-sheet during compression by PM-IRRAS and 2D IR correction methods [36,53]. In contrast, Bi and co-workers have reported that a series of model SP-C peptides in both DPPC and DPPG environments does not undergo their secondary structure interconversion from the α-helix into β-sheet but only keeps the hydrated α-helix upon compression [57]. Thus, the interpretation of secondary structure variation of proteins and peptides at the interface is still under development and controversial issue. In the present study, the interconversion into the β-sheet is not perfect but kept by ∼30% in both DPPG/Hel 13-5 and DPPC/DPPG/Hel 13-5 preparations due to the electrostatic interaction between Hel 13-5 and DPPG. This imperfection has been also reported by Cai and co-workers [33]. In the article, the KL4 peptide, which is a SP-B mimicking peptide with a net positive charge and is not an amphiphilic contrary to Hel 13-5, adopts a predominantly antiparallel β-sheet in the DPPC and DPPC/ DPPG mixtures. By contrast, KL4 highly keeps its secondary structure to the α-helix at all surface pressures in the DPPG/KL4 system. That is, the restraint to the β-sheet interconversion is accomplished in both DPPG/ cationic peptides systems. However, the restraint is even observed in the DPPC/DPPG/Hel 13-5, differently from the KL4 system. This might be attributed to the peptide feature (hydrophobicity, amphiphilicity,

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and amino acid sequence) and the magnitude of monolayer compression (maximum surface pressure of 45 mN m− 1 for the KL4 system). One of the significant functions in PS is a squeeze-out process, by which selective exclusions of the fluid components from monolayers take place at the collapse pressure of the fluid proteins to increase the surface concentration of DPPC during lateral compression [58,59]. Under the process, a surface reservoir made of the squeezed-out molecules is formed just below the monolayer and enables the molecules to respread to the interface upon subsequent expansion [4]. However, the respreading mechanism of PS components has not been made clear yet. The current PM-IRRAS result indicates the irreversible interconversion of Hel 13-5 secondary structures during compression and subsequent expansion. The KL4 peptide in the phospholipid mixtures also shows the irreversible interconversion [33]. On the other hand, Flach and coworkers have reported the reversible conversion from α-helix to β-sheet for a SP-B9-36 peptide in the binary DPPG system [60]. In the article, however, the maximum surface pressure just before film expansion is ∼27 mN m− 1, which is much lower compared with those for the KL4 system (∼ 45 mN m− 1) and own preparations (∼60 mN m− 1). The transition for the SP-B9-36 system may be induced by the insufficient heterogeneous intermolecular interaction and energy gain. For the clinical treatment to preterm infants with RDS, the administration of only one vial containing animal-based preparations (e.g., Survanta) shows a dramatic effect of treatment against them. The infants as such treated can gradually come to do a regular respiration on their own. Prior to the treatment, alveolar epithelial type II cells of the infants do not secrete pulmonary surfactants sufficiently. This is the cause of RDS for preterm infants. Therefore, the instillation of Survanta activates type II cell so that native pulmonary surfactants begin to be newly secreted from the cell. That is, pulmonary surfactant preparations firstly act as a temporary additive. Indeed, the irreversible interconversion of the peptide secondary structures may reduce pulmonary functions, and then the peptide would be metabolized to induce the secretion of new pulmonary surfactants. Herein, Hel 13-5 electrostatically interacts with the DPPG head group, when the mutual intermolecular distance becomes relatively small. The resultant surface reservoir of Hel 13-5 and DPPG is somewhat stable against respreading to the surface [19]. However, both components could respread toward the interface, when the mutual distance becomes larger upon expansion. Therefore, it is thought to be unlikely that the electrostatic interaction induces the irreversible interconversion of peptide secondary structures. This is also supported by the fact that the α-helix ratio for Hel 13-5 monolayers in the absence of phospholipids becomes somewhat smaller by a compression and expansion cycle (see Fig. S4 in the Supporting Material). Rather, the irreversibility may be related to the existence of the hydrated α-helix. The absorbance frequency of the α-helix marker band in Figs. 5–7 at 25 mN m− 1 during expansion is lower (∼ 1640 cm− 1) compared with the corresponding initial frequency (∼ 1650 cm− 1). This low frequency means that the structural transition to the hydrated α-helix is also the irreversible process. Therefore, it is speculated that compression of monolayers induces the interconversion of Hel 13-5 secondary structures from the α-helix to the β-sheet via the hydrated α-helix and that the interconversion is a reversible process. Namely, formation of the hydrated α-helix is stable at the interface and therefore may disturb the return to the original helix structure upon expansion. This interconversion process might be related to the formation of the surfaceassociated reservoir. Furthermore, as noted by Castano et al. [61], the present result supports the fact that the preservation of α-helix secondary structures during film compression and expansion is not an essential criterion for the surface activity of proteins or peptides containing charged and hydrophobic residues. In the present work, the pulmonary surfactant model preparations containing a mimicking peptide (Hel 13-5) of native surface lining SP-B

protein have been investigated to clarify the correlation between the Hel 13-5 secondary structure and the electrostatic interaction with phospholipids using the in situ PM-IRRAS technique. The current study has indicated that Hel 13-5 alone adopts mainly α-helical structure at the interface. In the presence of phospholipid monolayers, however, Hel 13-5 changes its conformation from the α-helix to the extended forms including the β-sheet during a compression and expansion cycle, and this interconversion is found to be an irreversible process. It is notable that the electrostatic interaction between charged moieties of DPPG and Hel 13-5 inhibits to some extent the interconversion to the β-sheet upon compression. Contrary to the reversible surface activity such as π–A isotherm hysteresis [19], the present spectroscopic data have exhibited the irreversible variation of Hel 13-5 secondary structure. This fact strongly suggests that the secondary structure is not necessarily related to the surface activity during respiration. Finally, it is noteworthy that surface biophysical techniques have made clear the fact of different behavior of protein or peptide secondary structures between the bulk and the monolayer for pulmonary functions, which provides deep insight into the design of model proteins and peptides for pulmonary surfactant replacement therapy and into the clarification of mechanism of native protein function near the alveolar surface. Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research 20500414, and Asian Core Program from the Japan Society for the Promotion of Science (JSPS). This work was also supported by a Grantin-Aid for Young Scientists 20810041 from JSPS (H.N.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbamem.2010.02.018. References [1] S. Schürch, J. Goerke, J.A. Clements, Direct determination of surface tension in the lung, Proc. Natl. Acad. Sci. USA 73 (1976) 4698–4702. [2] M. Amrein, A. von Nahmen, M. Sieber, A scanning force- and fluorescence light microscopy study of the structure and function of a model pulmonary surfactant, Eur. Biophys. J. 26 (1997) 349–357. [3] S. Krol, M. Ross, M. Sieber, S. Kunneke, H.-J. Galla, A. Janshoff, Formation of threedimensional protein–lipid aggregates in monolayer films induced by surfactant protein B, Biophys. J. 79 (2000) 904–918. [4] D.Y. Takamoto, M.M. Lipp, A. Von Nahmen, K.Y.C. Lee, A.J. Waring, J.A. Zasadzinski, Interaction of lung surfactant proteins with anionic phospholipids, Biophys. J. 81 (2001) 153–169. [5] H. Nakahara, S. Lee, G. Sugihara, O. Shibata, Mode of interaction of hydrophobic amphiphilic α-helical peptide/dipalmitoylphosphatidylcholine with phosphatidylglycerol or palmitic acid at the air–water interface, Langmuir 22 (2006) 5792–5803. [6] S. Schürch, R. Qanbar, H. Bachofen, F. Possmayer, The surface-associated surfactant reservoir in the alveolar lining, Biol. Neonate 67 (Suppl 1) (1995) 61–76. [7] S.-H. Yu, F. Possmayer, Lipid compositional analysis of pulmonary surfactant monolayers and monolayer-associated reservoirs, J. Lipid Res. 44 (2003) 621–629. [8] P. Krüger, J.E. Baatz, R.A. Dluhy, M. Lösche, Effect of hydrophobic surfactant protein SP-C on binary phospholipid monolayers. Molecular machinery at the air/ water interface, Biophys. Chem. 99 (2002) 209–228. [9] R. Veldhuizen, K. Nag, S. Orgeig, F. Possmayer, The role of lipids in pulmonary surfactant, Biochim. Biophys. Acta 1408 (1998) 90–108. [10] J.C. Clark, S.E. Wert, C.J. Bachurski, M.T. Stahlman, B.R. Stripp, T.E. Weaver, J.A. Whitsett, Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice, Proc. Natl. Acad. Sci. USA 92 (1995) 7794–7798. [11] L.M. Nogee, G. Garnier, H.C. Dietz, L. Singer, A.M. Murphy, D.E. deMello, H.R. Colten, A mutation in the surfactant protein B gene responsible for fatal neonatal respiratory disease in multiple kindreds, J. Clin. Invest. 93 (1994) 1860–1863. [12] P.L. Ballard, L.M. Nogee, M.F. Beers, R.A. Ballard, B.C. Planer, L. Polk, D.E. deMello, M.A. Moxley, W.J. Longmore, Partial deficiency of surfactant protein B in an infant with chronic lung disease, Pediatrics 96 (1995) 1046–1052. [13] J. Pérez-Gil, C. Casals, D. Marsh, Interactions of hydrophobic lung surfactant proteins SP-B and SP-C with dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol bilayers studied by electron spin resonance spectroscopy, Biochemistry 34 (1995) 3964–3971.

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