Probing Intermolecular Interactions in Phospholipid Bilayers by Far

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Probing Intermolecular Interactions in Phospholipid Bilayers by FarInfrared Spectroscopy Giovanna D’Angelo,*,† Valeria Conti Nibali,‡ Cristina Crupi,† Simona Rifici,† Ulderico Wanderlingh,† Alessandro Paciaroni,§ Francesco Sacchetti,§ and Caterina Branca† †

Dipartimento di Fisica e Scienze della Terra, Università degli Studi di Messina, 98122 Messina, Italy Institute for Physical Chemistry II, Ruhr-University Bochum, 44801 Bochum, Germany § Dipartimento di Fisica, Università degli Studi di Perugia, 06123 Perugia, Italy ‡

S Supporting Information *

ABSTRACT: Fast thermal fluctuations and low frequency phonon modes are thought to play a part in the dynamic mechanisms of many important biological functions in cell membranes. Here we report a detailed far-infrared study of the molecular subpicosecond motions of phospholipid bilayers at various hydrations. We show that these systems sustain several low frequency collective modes and deduce that they arise from vibrations of different lipids interacting through intermolecular van der Waals forces. Furthermore, we observe that the low frequency vibrations of lipid membrane have strong similarities with the subpicosecond motions of liquid water and suggest that resonance mechanisms are an important element to the dynamics coupling between membranes and their hydration water.

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INTRODUCTION Despite the intense study to understand the molecular dynamics mechanisms that underlay the biologically relevant processes in lipid membranes, this issue is in need of additional research. In particular, the role of pico- and subpicosecond dynamics in activating important biological functions in membranes remains an important question to be answered. While these time scales are difficult to probe experimentally, it is becoming increasingly clear that the collective thermal fluctuations of the lipids control the passive transport of molecules through cell membrane.1 In the past years many efforts were devoted to gather information on the incoherent (single-particle) thermal motions of phospholipid model membranes2−7 and on the dynamics of water molecules at the interface with membrane.8−11 Lately, the interest of many investigations moved to the study of the fast picosecond coherent (collective) dynamics on the mesoscopic scale, which corresponds to propagating, phonon-like dynamics.14−19 Phonon-like excitations can drive the density fluctuations in coherence favoring ultra long-range interactions of much larger extent than that of chemical forces.12,13 As a result, analogously to what is proposed for proteins,20,21 they can be expected to play a main role in many biological membrane processes of strong relevance, especially in those involving the transfer of energy. Recently, the vibrational collective dynamics of lipid bilayers was investigated by MD simulations.22 This study revealed the existence of a transverse acoustic-like phonon mode in addition to the known longitudinal one14,16,17 and showed that these © XXXX American Chemical Society

systems support several low energy optical-like phonon modes in the spectral region below 35 meV as well. The high-energy optical vibrational modes are potentially involved in the vibrational energy transfer and in the hydrogen-bond dynamics at the membrane-water interfaces.23,24 So the need for their experimental evidence and accurate identification is mandatory in view of the related biological implications. So far the experimental studies investigating the coherent vibrational dynamics of lipid multibilayers,1,14,16 although they confirmed some of the simulation predictions, had poor success in revealing the fine details of the high energy (>10 meV) collective excitations. In fact the application of inelastic X-ray and neutron scattering techniques is restricted by the high degree of static and dynamic disorder of the biological systems, that, by reducing the timelife of the excitations, makes particularly difficult the precise discernment of the high energy modes.1 In addition all the experimental studies of fast subpicosecond coherent dynamics in lipid membranes have been performed on fully hydrated samples.14,16,19 As a result, no experimental information is yet available about the subpicosecond dynamics of phosholipid membranes in dry or low water content condition. Here we report the results of an experimental study of the far-infrared region on DMPC stack bilayers aimed to verify the Received: October 12, 2016 Revised: December 12, 2016 Published: January 24, 2017 A

DOI: 10.1021/acs.jpcb.6b10323 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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the intermediate hydration states was not possible, due to continuously changing extension of the evanescent wave into the sample during dehydration (see the Supporting Information for experimental details), we were able to identify the fully dehydrated sample, basing on the observation of the disappearance of the water vibrational bands in the spectrum. This is shown in Figure 1 for the OD stretching region, where it

existence of the optical modes predicted by our previous MD study22 and to identify them. The infrared spectra were collected while the sample was progressively dehydrated in an attempt to unravel the intrinsic vibrations of lipid membrane and to catch the details of the acyl chain motion in the anhydrous bilayer, useful for understanding of the fully hydrated functional state of membrane.

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EXPERIMENTAL DETAILS AND DATA ANALYSIS Preparation of Sample. Aligned multilayers of DMPC (Avanti Polar Lipids) were obtained by following a procedure reported elsewhere.25 Briefly, the lipids were dissolved in an excess of [2:1] CHCl3/CH3OH (chloroform/methanol) solution. Then the lipid solution was dried and finally for each milligram of lipid it was redissolved in 15 μL 2:1 CHCl3/ CH3OH containing a [1:1] molar ratio of naphthalene to lipid. To remove the naphthalene and any residual organic solvent, the samples were vacuum-dried overnight. Once the sample was placed on the ATR crystal, the lipid multilayers were hydrated by keeping them under a cover for volatile samples for 12 days in atmosphere with 96% relative humidity using a saturated potassium sulfate D2O solution. Then, 36 mol of D2O per mole of lipid were dropped on the sample in order to obtain a fully hydrated sample. Note that in this study we chose to hydrate the membrane with deuterated water in order to make the data directly comparable with the results of our recent Brillouin neutron scattering study on the same system (unpublished results). FTIR-ATR Measurements. Far and mid-IR absorbance spectra were recorded under vacuum at 26 °C with a diamond crystal ATR (attenuated total reflectance) mounted on a Vertex 80 V FT-IR spectrometer (Bruker) (see Supporting Information for further details). A background scan was recorded prior to the measurement and subtracted from the sample spectra. Each spectrum was averaged over 128 scans with a resolution of 2 cm−1 and ATR corrected. The experiment started by collecting the spectrum of the full hydrated sample. Then, after removing the cover, sets of IR spectra were collected under vacuum at interval of 60 s for 2 h and of half an hour for the rest of the experiment. The experiment was repeated three times with similar results (see the Supporting Information) All the far-IR spectra were decomposed into Gaussian components using standard algorithms, leaving all fit parameters free to vary (95% confidence interval). The optimal number of components was determined through a careful statistical study, as described in the text and in the Supporting Information.

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Figure 1. Mid-IR spectra of: (bottom) fully hydrated DMPC sample (black dot dashed line); (top) the same sample at two distinct dehydration times: “72 h” (red dashed line) and “170 h” (blue solid line) (h indicates hours). Shading indicates the OD and CH stretching regions of water and lipid acyl chains, respectively.

is clear that the water broad peak around 2500 cm−1 vanishes keeping the sample under vacuum for at least 3 days. Moreover, from this figure it is evident that the spectrum acquired after 72 h differs not significantly from that acquired after 1 week. This observation, together with the disappearance of the water peak, provides indication for the full dehydration of the sample. Therefore, in the following we will refer to the spectrum after 72 h as the spectrum of the dry sample. As an additional check for the hydration level and to verify the phase state of bilayers, a careful study of the whole midinfrared region was carried out. As known, the morphological and phase state properties of lipid multilayers are strongly sensible to the presence of water: when the water content decreases, the main transition temperature is shifted to higher values, the area per polar head decreases, whereas the order among the hydrocarbon chains increases (this order is signed by the change from trans to gauche conformations of the hydrocarbon chains of the lipid). In addition the disappearance of free water between the bilayers leads to a decrease of the lamellar repeat distances.27 As a result the van der Waals interactions among the hydrocarbon chains become more efficient and the dried multibilayers are in gel phase at temperatures at which they would be in liquid crystalline phase if they were hydrated. By analyzing the FTIR spectra in the mid-infrared region we could follow the lyotropic lipid phase transitions and the structural conformations induced by the removal of water. In fact phospholipids contain molecular groups located in different regions of the bilayers, whose related absorption bands are sensitive to structural and morphological changes as well to environmental conditions causing them.28 Upon progressive dehydration, we observed that many of the bands in the low and mid-infrared regions changed in shape, position, and intensity, providing us information on the expected characteristic changes in the lipid-packing density and of the lipid

RESULTS AND DISCUSSION

The predominant features of the vibrational spectra of hydrated phospholipid multibilayers in far-IR region (30−300 cm−1) are generally ascribed to the torsional modes of the hydrocarbon chains, skeleton vibrations, and to the motions arising from intermolecular hydrogen bonds between water−water, water− lipid, and lipid−lipid molecules.26 These collective modes provide a singular fingerprint of the conformational states of the phospholipids in membranes and are expected to be sensitive to water content changes. To control the level of hydration of the sample, we monitored the decrease of the absorbance in the mid-IR region of both the OD-stretching (2500 cm−1) and DOD-bending (1209 cm−1) vibrational bands of water. Although an exact quantitative estimate of water content in B

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Figure 2. (a) Top, far-IR spectrum of D2O; bottom, far-IR spectra of DMPC/D2O sample at four distinct dehydration times (see text for details): H0 (blue square), H1 (green down-triangle), H2 (red up-triangle), and H3 (black circle). In (b) the same spectra in (a, bottom) are shown separately together with the corresponding fitting curve (solid black line) obtained as the sum of the individual Gaussian peaks (solid colored lines).

Table 1. Summary of the Frequencies and Tentative Assignments of Bands Identified in This Work, Compared with the Data from Ref 25 and the Data on Bulk Water36,37 absorption bands

wet DMPC frequency cm−1

dry DMPC frequency cm−1

A B C D E F G H

50 70 89 116 155 191 217 260

50 70 89 110 136 186 221 258

tentative assignment van der Waals van der Waals van der Waals C−CH3 motion14,17 van der Waals τ(C − CH2) τ(C − CH3)

DMPC from ref25 frequency cm−1

liquid water frequency cm−1 50−6037

96 hydrogen bonds

8936

189 τ(C − CH2)

12936 19336

251 τ(C − CH3)

25036

From Figure 2a it clearly appears that the infrared absorbance in the far-IR region decreases as the dehydration proceeds. In particular, in the dry sample, H3, we observe a strong broad band at about 90 cm−1 and three less intense bands above 150 cm−1 (Figure 2b). These spectral features are present also in the hydrated samples, but they become less and less discernible and progressively badly defined as water content increases. Note that the spectrum of the full hydrated bilayers, H0 sample, although in some way resembles that of D2O (see Figure 2a, top), is quite different from it. The apparent asymmetric shape and the composite profile of the first band in the H3 spectrum led us to think that this band is more structured than what appears at first glance. To quantitatively analyze the experimental data we performed curve fitting of the infrared spectrum as a linear combination of individual Gaussian component bands by the iterative adjustment of their width, position and relative weights (fraction of the total peak area). The process was iterated until an acceptable fit between the computed and experimental spectrum was obtained. Particular attention was paid to obtain the best possible fit with the minimum number of component bands. Initial peak positions were determined from a visual inspection of the spectrum. Thus, the initial fit started with four individual bands; then the spectral regions showing the larger residuals, with respect to the fitting curve, were examined visually and additional components were added as appropriate

conformations related to the drying-promoted phase transition from liquid to solid-gel.28,29 We have monitored the morphological changes that accompany the dehydration of the membrane in the interface water/lipid region by observing the frequency shifting of the infrared peak associated with the asymmetrical stretching mode of PO. This mode is highly sensible to water, and involved in the formation of H-bonds with water molecules. Its related infrared peak was observed to shift progressively from 1260 to 1232 cm−1 (data not shown), as expected for dehydrated and fully hydrated multilayers, respectively.28,30−32 Moreover we have controlled the chain order of the hydrophobic interior of lipid bilayers by analyzing the vibrational frequency of the symmetric CH2 stretching mode at around 2850 cm−1. The frequency of this band changes according to acyl chain conformation.28,30,31 Upon dehydration, we found that it shifted from 2852 cm−1 (fully hydrated sample) to 2850 cm−1 (dried sample) (data not shown), indicating the transition of the lipid bilayers from a liquid crystalline to a gel state.28,30−32 The dehydration process caused more marked changes in the far-infrared region: four representative spectra taken at different times 0, 2, 9, and 72 h (labeled in the following H0, H1, H2, and H3, respectively) corresponding to four progressively decreasing hydration levels of the sample were chosen and reported in Figure 2a. C

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the water is dried off. The faster reduction in fwhm was observed for the modes E, F, and H. Since the lipids are in the liquid (disordered) phase when fully hydrated, whereas they are in the solid (ordered) gel phase when dehydrated, we infer that the observed decrease of the FWHMs mainly marks the gradual morphological changes of the membrane through more and more ordered phases which are accompanied by the decreased motional (chain flexing and twisting) rates and by a smaller number of conformational states of the hydrocarbon in the gel phase. Thus, the same vibrational modes characterize the different phases of DMPC, but their vibrational width is influenced by the topological or dynamic disorder of the specific phase at which the system is: higher disorder corresponds to higher width of vibrations. On the other hand the decrease of the peak intensity and the faster decrease of fwhm with dehydration observed for the bands E, F, and H is believed to reflect the average loss of water−water hydrogen bond interactions of the interlayer water. As concerning the microscopic origin for the identified vibrations, we attribute the bands F and H to the torsion modes of the hydrocarbon chains and terminal methyl groups, respectively.26 Furthermore, on the basis of the similarity of their characteristic energies, we are tempted to identify the band D with the non dispersive mode predicted by Tarek et al.17 and observed by neutron scattering,14 and to attribute it to a unspecified motion of terminal (methyl) carbons of the chains. In contrast with the previous result,17 we observed that this peak shifts toward lower wave numbers when the membrane undergoes a transition from liquid to gel phase upon dehydration, suggesting a hindered mobility of the related molecular groups. Furthermore, we propose a different origin for the bands at A, C, and E. In phospholipid bilayers these bands are generally considered as a unique broad band originated from the molecular breathing and from the intermolecular hydrogenbonding structure within the phospholipid bilayers.26 Intermolecular hydrogen bonds in biological membrane lipids can be usually formed between the lipids and interfacial water or between neighboring lipid molecules. But, in the DMPC lipid bilayers here investigated, the phospholipids can form hydrogen bonds exclusively with water (through the nonesterified oxygen of lipid head groups).33,34 Thus, the presence of these bands even in the fully dried system, implies that the related vibrations do not involve hydrogen bonding interactions. We suggest that weak intermolecular interactions (unbonded interactions) like dispersive and dipole−dipole forces are responsible for these modes. These interactions are, in fact, characterized by force constants smaller than those for hydrogen bonding, and by vibrational energies typically less than 150 cm−1.35 These forces are consolidated in the gel state due to the approach of the hydrocarbon chains and the increase of their order. In particular we consider the observed gradual increase of the intensity of the bands B, C, and D and the shift to lower wavenumber of the band D upon dehydration as an indication of increased cooperative effects according to which more strong interchain bonds are formed. It is important to remark that, because the width of the contributing Gaussians is greater than the separation between the maxima of adjacent peaks, the fitting of the infrared spectra to Gaussians whose widths can change could open up questions on the uniqueness of the fit. However, the consistence between

(see Figure S2). The minimum number of Gaussians to retain for the best analysis of the spectra was determined by evaluating the number of components that minimize the residual sum of squares (RSS). In this way, we found that eight components were needed to fit accurately the observed profile for the H3 sample (Figure S3, top-left) and that the contour of the first band was composed of five main sub-bands. (Figure S2c). Then, considering that the eight vibrations found for the dry lipid must be reasonably present in the wet samples, we started the curve fitting iteration for the H0, H1, and H2 spectra with the same eight components of H3. Although this procedure produced very good fits, also for these samples a complete study of the RSS behavior by varying the number of the Gaussians was done. Interestingly, we noted that the same number of eight components was necessary to reproduce the spectral profile below 300 cm−1 for all the samples (see Figures S3 and 2b). This finding indicates that, whatever the hydration level is and the consequent structural (liquid, ripple or solid-gel) or morphological phase of the lipid bilayer, the phospholipids sustain the same low energy vibrational modes. These vibrations in the fully hydrated sample, H0, are centered at 50, 70, 89, 116, 155, 191, 217, and 260 cm−1. In the following the corresponding bands will be labeled with A, B, C, D, E, F, G, H, respectively, as reported in Table 1. Moreover the maximum frequency of the modes D, E, F, and G was observed to shift during dehydration, showing high sensitivity to the presence of water. Conversely, the frequency of the other modes did not change appreciably, thus indicating that the associated vibrations arise from collective motions of molecular groups that do not interact with water molecules. We found also that, although a general decrease of the total absorbance in the region below 300 cm−1 is observed upon dehydration, the percentage areas of the individual bands (calculated from the area of each peak compared to the total integrated area) show different trends: while the area of the bands B, C, and D increases, reaching a saturation after about 60 h, the intensity of all the other bands decreases more or less (as in the case of the band A) markedly (see Figure 3). In the same figure we also show the dependence of the fullwidth half-maximum (fwhm) values of the eight bands vs the dehydration time. For all the components, fwhm decreases as

Figure 3. Dependence on the dehydration time of the full-width halfmaximum (fwhm) values (empty square) and of the percentage area(full square) of the eight band components obtained by the fitting procedure. D

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proved to control the passive transport of molecules through cell membrane.1 Moreover, due to their long-range correlation character, it is also reasonable to believe that these low energy modes play a key role in tailoring the interaction with solvent. Concerning the latter issue, it is worth noting that the frequencies of bands C, E, F, and H in the dry sample are very close to the frequencies of modes that generate the so-called connectivity band in liquid bulk water (89, 129, 193, and 250 cm−1),36 that are ascribed to collective intermolecular stretching modes of the hydrogen bonds (HBs). The frequency of the band A is close to the frequency of the peak at 50−60 cm−1 revealed in the Raman spectra,37 and ascribed to transverse acoustic (TA) standing waves (H-bond bending restricted translation (perpendicular to O−H···O)). For convenience, the frequencies of the water modes together with all the other frequencies discussed in this article are summarized in Table 1. The superposition between the low-frequency collective vibrational modes in bilayers and those contributing to the connectivity band in liquid water26 suggests that there could be an effective dynamical coupling between DMPC and solvent. We can speculate that these low-energy resonances are at the origin of the long-range interactions between membrane and water, i.e., the landscape of collective fluctuations sustained by lipid bilayers is intrinsically able to resonate together with water. This coherent motion may be relevant for the energy transfer and signaling between cells. A similar dynamic coupling has been proposed to exist between proteins and water.39,40

Figure 4. Left: Gaussian deconvolution of the far-IR spectrum (open symbols) of dehydrated DMPC (H3). The horizontal dotted lines indicate the energies of the peaks obtained by Gaussian fitting. Right: the dispersion curve of the transverse acoustic-like (red solid line), the longitudinal acoustic-like (black solid line), and the optic-like modes (all other symbols) from MD simulations of the DMPC gel phase.22 The cross symbols indicate the energies of the excitations revealed by MD extrapolated to Q = 0 Å−1.

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phase obtained by MD simulations.22 To compare the data, the wavenumbers were converted to meV. Importantly, we observe that the energy values of the optical phonon branches revealed by the MD analysis extrapolated to the zero-momentum of the Brillouin zone (indicated by cross symbols in the Figure 4) agree very well with the energy values of the IR modes, marked as dashed lines in the same figure. This finding supports experimental evidence for the existence of all the optical modes predicted by our previous MD study22 and proves that these modes are infrared active. Thus, in the far-IR range, lipid bilayers have a rich spectrum of low frequency collective modes, that arise mainly from the coupling of vibrations of different lipids through intermolecular van der Waals forces. We remark that the existence in lipid bilayers of a so high number of vibrations below 300 cm−1 is a indication of high side-chain and main-chain conformational fluctuations. These fluctuations provide a physical mechanism by means acyl chains can organize and spatially segregate membrane components. Moreover, the fact that the low energy vibrations remain almost unchanged when the acyl chains undergo the transition from gel to liquid phase suggests that these modes are rather isolated from other degrees of freedom. This observation leads to the conjecture that energy (the thermal energy and/or the energy released from the binding of protein or enzyme to the membrane surface) might stay localized and stored in these modes. This energy can be used to modulate the elastic properties of the lipid bilayer and promote functional properties, such as diffusion and transport through the membrane, or can be relevant for transmembrane protein functions.38 In this regard, the low energy phononic motion of the hydrocarbon tails in phospholipid membranes was recently

CONCLUSIONS

This study revealed that the far-IR spectra of the DMPC bilayers contain more bands in the subpicosecond region than previously detected26 and indicated that van der Waals intermolecular vibrations prevail on the intermolecular hydrogen bond vibrations. Furthermore, although both the environment and even more the hydration of the phospholipids are known to strongly affect many dynamical properties of the bilayers, the present results show that the picosecond and subpicosecond vibrational dynamics of the membrane are not so influenced by the phase state of the lipids and by the hydration. Our results demonstrate that a careful analysis of the farinfrared region as a function of hydration is an efficient tool for unravelling the structure of the connectivity band of lipid bilayer and potentially of other biomolecules. Additionally we revealed strong similarities between the THz dynamics of lipid membranes and those of liquid water as in the case of dry and hydrated proteins.39−41 In summary, this work is a first step toward a better understanding of the molecular vibrations of phospholipid bilayers in the THz frequency range. We hope that the present results will stimulate new experimental and theoretical investigations of acoustic modes of lipid bilayers to understand in detail what motions correspond to these low energy modes, what are the consequences of these collective vibrational motions for the dynamics of hydration, and finally what is their role in the functions of biological membrane. E

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b10323. Detailed description of the sample preparation, experimental methods, and data analysis (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Giovanna D’Angelo: 0000-0002-3548-3255 Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.jpcb.6b10323 J. Phys. Chem. B XXXX, XXX, XXX−XXX