Temperature-Dependent Hydrocarbon Chain Disorder in

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Temperature Dependent Hydrocarbon Chain Disorder in Phosphatidylcholine Bilayers Studied by Raman Spectroscopy Alexey A. Dmitriev, and Nikolay Vladimirovich Surovtsev J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b07502 • Publication Date (Web): 26 Nov 2015 Downloaded from http://pubs.acs.org on December 9, 2015

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The Journal of Physical Chemistry

Temperature Dependent Hydrocarbon Chain Disorder in Phosphatidylcholine Bilayers Studied by Raman Spectroscopy

A. A. Dmitriev,1,2,3 and N. V. Surovtsev,*1,2 1

Institute of Automation and Electrometry, Russian Academy of Sciences, Novosibirsk, 630090, Russia, 2

Novosibirsk State University, Novosibirsk, 630090, Russia,

3

Institute of Chemical Kinetics and Combustion, Russian Academy of Sciences, Novosibirsk, 630090, Russia.

E-mail address: [email protected] Fax: (7)-3833-333863

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Abstract The Raman scattering of five phosphatidylcholines (1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine

(POPC),

1,2-dihexadecanoyl-sn-glycero-3-phosphocholine

dioleoyl-sn-glycero-3-phosphocholine

(DOPC),

(DPPC),

1,2-

1,2-dilinoleoyl-sn-glycero-3-phosphocholine

(DLPC) and 1,2-dilignoceroyl-sn-glycero-3-phosphocholine) was studied in a wide temperature range. These phospholipid bilayers are different in temperature of the gel-fluid transition Tm (from – 57 oC to +80 oC) and in number of the unsaturated bonds. For all lipids the temperature dependences of both asymmetrical methylene stretching and C-C stretching bands evidence that the disordering processes occur significantly below Tm. Temperature onset of the decrease of the Raman intensity of the asymmetrical methylene stretching band is the same for the unsaturated lipids (DLPC, POPC and DOPC), that was interpreted as the importance of the packing defects in the bilayers of these phospholipids. The chain conformational order was characterized by the Raman intensity of the high-frequency C-C stretching mode. An approach was used where the Raman intensity of this mode serves as a measure of the hydrocarbon chains in the ground conformational state. The temperature dependence of the chains in the ground conformational state was well described by a simple model with the ground and two excited states, being the kink and the highly disordered, fluid-like, state.

Keywords: phospholipid bilayers, Raman scattering, conformation, gel-fluid transition.


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Introduction Phospholipid bilayer is a matter of intensive study for a long time. Despite of the relatively simple structure of the bilayers there are a lot of topics in chemistry and physics of phospholipid bilayers, which are not fully understood. The reason is a complex interplay between different degrees of freedoms of the bilayers: conformations of hydrocarbon chains, lateral packing, interchain interaction, librational motion of chains, anharmonical motion of hydrogen atoms, headgroup interaction and others. The most striking phenomenon in lipid bilayers is the phase transition from the ordered gel state to the disordered fluid (liquid crystalline) state.1 This transition in lipid bilayers attracts the attention due to its biological relevance and its importance for the bilayer application in drug delivery systems. A lot of experimental and theoretical works were devoted to study the order-disorder changes during the gel-fluid transition. Disordering processes in bilayers during the gel-fluid transition are characterized by various structural2,3 and spectroscopic experimental techniques, including NMR,4-6 infrared absorption,2,4,7-11 electron spin resonance,6,12-15 Raman scattering,16-19 Brillouin light scattering,19 calorimetry6,20 and computer simulation.21,22 Usually the studies of disordering processes are restricted in the temperature range near the gel-fluid phase transition temperature (Tm), implying that the bilayer structure is the same below the Tm. Indeed, results of structural and calorimetric techniques do not reveal significant changes below the Tm (except for transitions to ripple and subgel phases that are sometimes detected near the Tm).23 However, there are experimental indications that the disordering processes occur also at temperatures significantly below Tm.24,25 Knowledge about the lowtemperature disordering processes is important for fundamental topics of physics and chemistry of phospholipid bilayers as well as for some applications using the effect of passive permeation across a lipid membrane. In previous works the capability of the Raman spectroscopy to study the disordering processes below Tm was demonstrated, where the sensitivity of the 2880 cm-1 antisymmetric CH2-stretching vibration line to intermolecular packing interactions26 and the sensitivity of the intensity of the 1130 cm-1 line (C–C stretching vibration) to chain conformations27,28 were used. In the case of the DPPC bilayers (saturated hydrocarbon chains) the disordering processes observed even at ~150-230 K, while Tm = 314 K.26,27 It was found that in DPPC and monounsaturated POPC bilayers28,29 the conformational statistics can be well described by a two-excited-state model, which consider only two excited states of the hydrocarbon chains: the kink state and highly disordered, “melted”, state. These studies reveal a potential of the Raman spectroscopy plus the two-excited-state model in description of conformational statistics of hydrocarbon chains. However, it is not clear whether results found for POPC and DPPC can be 3 ACS Paragon Plus Environment


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extended to other lipids with significantly different Tm, or with two unsaturated bonds? Is the excitation energy of the melted state has a counterpart in the enthalpy of the gel-fluid transition? These and other questions need further investigations over a set of different phospholipids bilayers. Here, by Raman spectroscopy we studied the bilayers of five phosphatidylcholines, which are different in Tm (from – 57 oC to +80 oC) and in number of the unsaturated bonds. Both asymmetrical methylene stretching and C-C stretching bands evidence that the disordering processes begin at temperatures significantly below Tm. Enthalpy of the gel-fluid transition was found by differential scanning calorimetry. The conformational statistics of hydrocarbon chains was described by a modified two-excited-state model.

Experiment Sample preparation. Five phospholipids were used in the work: 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC), 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-dilignoceroyl-sn-glycero-3-phosphocholine (PC24). All of them were purchased from Avanti Polar Lipids (purity > 99%) and used without further purification. Chemical formulas of the phospholipids are presented in Figure 1 together with the main transition temperatures reported in the products webpage.30 All of phospholipids have the same polar head, but they are different in the hydrocarbon chain length and in the number of unsaturated C–C bonds. The samples of different lipids were prepared using following procedures. At first the phospholipids powder was hydrated in distilled water at temperature above the gel-to-fuid phase transition temperature. Then mixture undergone several (five-six) cycles of cooling and heating when the sample passed through the ice formation and the phase transition temperatures. After that the samples were centrifuged for 5 min at 20-40 °C with 10000 rev/min and the excess of water was removed. This protocol provides the aqueous suspension of multilamellar lipid vesicles. The final lipid-to-water concentration was typically ~1:1.5 by weight. For some samples this ratio was somehow higher (up to 1:4), depending on particular details of treatment. Optical microscopy reveals that these procedures lead to spherical multilamellar vesicles with diameters from 1 to 5 µm. Number of phospholipids bilayers within one vesicle was estimated in the range 101-102. Variations of parameters of the preparation procedure change vesicle parameters (average diameter, number of bilayers and others), but no effects for Raman data, discussed in the present work, were found. For the purpose of Raman spectroscopy the prepared samples were placed into the glass ampoules and sealed. 4 ACS Paragon Plus Environment

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Differential scanning calorimetry (DSC). Samples of the aqueous suspension of lipid vesicles were loaded into a standard aluminum crucible of the DSC-200F3 Netzsch and cooled for temperatures below the ice formation and main transition temperatures. The heating curves through the main transition and ice melting temperatures were determined with a heating rate of 2 K/min. In the case of the POPC bilayer the cooling was used in the DSC experiment to resolve the lipid main transition from water-to-ice transition. The peaks of the DSC curves provided the gel-to-fluid transition temperature Tm and the transition enthalpy ∆H. Raman spectroscopy. Raman spectra of opaque white samples were measured at a nominally right scattering angle using a triple-grating TriVista 777 spectrometer and a 532-nm solid-state laser. Spectral ranges 675-1170 cm-1 and 2750-3120 cm-1 were studied. Other details of the Raman experiment were the same as previously reported.27 The sample was placed into an optical closed-cycle helium cryostat for measurements at low temperatures. The temperature range covered from 50 K to 320 K in the case of DLPC, DOPC, POPC and DPPC. The sample was firstly cooled to the lowest temperature and then heated to the temperature of the Raman spectrum acquisition. In case of PC24 the low-temperature measurements were accomplished by the Raman experiment in a furnace, covering the temperature range from 300 K to 366 K. Results The values of Tm and ∆H found in the DSC experiment are presented in the Table. The values of the main transition temperature are in good agreement with the values from the products webpage (Figure 1). The main uncertainty in the ∆H values (Table 1) is caused by the uncertainty lipid-to-water weight ratio in the sample preparation. The uncertainty in the ∆H values was estimated by comparing the results from different samples. In the case of DLPC the DSC peak corresponding to the main transition is weak, resulting in an additional contribution to the uncertainty. Our data for ∆H values are in the reasonable agreement with the ∆H values reported in literature for DPPC,4,31-39 POPC,31,40-45 DOPC,46-50 DLPC,51 and PC24.39 Figure 2 illustrates the representative Raman spectra in the spectral ranges of the C–C and C–N stretch vibrations for the PC24, DLPC and DOPC bilayers. The Raman spectra for DPPC and POPC bilayers were similar to those presented in refs. [27,28]. As in refs. [27-29] the Raman mode near 720 cm-1, corresponding to C-N stretching vibration, was used as the reference mode, whose integral intensity is temperature independent according to ref 52 (and reference therein). In the spectral range 1000-1180 cm-1 (Figure 2) the contribution from the skeletal mode vibrations (C-C stretch) of the hydrocarbon chains dominates. 5 ACS Paragon Plus Environment


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It is seen that when temperature is changed the Raman intensities redistribute among the C-C vibration lines (Figure 2). This redistribution is related to the change of the conformational states of the hydrocarbon chains with temperature. Especially strong changes in the Raman intensities of the C-C vibrations occur at the gel-fluid transition (Figure 2). There is a general tendency that the sharp lines of the gel phase transform to the featureless band of the fluid phase. It was suggested27 that for the analysis of the chain conformational states it is convenient to use the mode near 1130 cm-1, which was ascribed to the all-trans conformational state of the DPPC hydrocarbon chains. Indeed, the theoretical analysis53 evidences that the sections of the lipid containing more than 10 trans bonds contribute to the 1130 cm-1 line of the DPPC bilayer. Despite the fact that the attribution of the 1130 cm-1 line intensity solely to the all-trans state somehow overestimates the concentration of the all-trans state lipid chains, early this approach was found to be useful in applications for the DPPC and POPC bilayers.27-29 This approach cannot be extended to the unsaturated phospholipids DOPC and DLPC, whose chain conformations cannot be in all-trans state. Let’s suggest that in the low-temperature limit the phospholipid chains occupy a most favorable energy state and that this low-lying (ground) state has maximal number of trans conformations. The conformational states with maximal number of trans conformations is expected to be characterized by the highest frequency among the C-C vibrations.53 In the case of the DPPC and POPC bilayers the ground state can be characterized by the mode near 1130 cm-1, and the similar mode is seen near 1125 cm-1 in the case of the DOPC bilayer (Figure 2c). Peculiarity of PC24 chain packing lead to splitting the peak near 1130 cm-1 into three components (1110, 1130, and 1170 cm-1) in the low-temperature gel phase (Figure 2a, more about different gel-state phases of long-chain phospholipids see in ref 8). It is convenient to follow the behavior of the 1130 cm-1 line as the representative of the highest frequency modes of the C-C vibrations in the case of PC24. In the case of the DLPC bilayer the highest frequency mode is near 1115 cm-1 at low temperatures (Figure 2b). From Figure 2b it is seen that this mode behaves similarly to the highest frequency modes of other phospholipids. Thus, the intensity of the 1115 cm-1 mode can serve as a measure of the number of DLPC hydrocarbon chains in the ground state. We will use the notation IntCC for the integral intensity of the highest frequency C-C mode (the line at frequency of 1130 cm-1 in the case of DPPC, POPC, PC24, of 1125 cm-1 in the case of DOPC, and of 1115 cm-1 in the case of DLPC). To quantify the temperature dependence of the highest frequency modes the integral intensities of the highest frequency C-C mode and of the C-N stretching mode (IntCN) were found from the experimental Raman spectra. For this purpose the C-N stretching mode was described by a Gaussian contour and the experimental spectrum in the range of 1030-1150 cm-1 by a sum of few (from three to five depending on a particular lipid) Lorentzians. The temperature 6 ACS Paragon Plus Environment

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dependences of the ratio IntCC/IntCN for all phospholipids are shown in Figure 3a. Raman intensity IntCC reflects the number of C-C vibrational modes with high frequency and the modulation of the polarizability tensor (Raman tensor) by these modes.54 In the framework of the assumption that in the low-temperature limit all phospholipids take the ground state, the value of IntCC at T→ 0 K reflects the modulation of the polarizability tensor by a particular ground state, which is different for different phospholipids. The magnitude of IntCC/IntCN in the low-temperature limit is different for different lipids, revealing the differences in the ground conformational state. In case of the saturated lipids (DPPC and PC24) the polarizability increases with the number of C-C bond in hydrocarbon tails. It is notably that the low-temperature values of IntCC/IntCN are very close for the DOPC and POPC bilayers. This suggests that the saturated chain of POPC takes the conformation similar to one of the monounsaturated chain. Reducing the number of chains in the ground state with the temperature increase is seen in Figure 3a. This reduction is related to the occupation of the conformational states with higher energy. To consider the temperature dependence of this occupation it is convenient to normalize IntCC/IntCN by its lowest-temperature value. We considered the scaled ratio of IntCC/IntCN RCC (T ) =

IntCC (T ) / IntCN (T ) IntCC (T = 0 ) / IntCN (T = 0)

(1)

as a measure for the concentration of hydrocarbon chains in the ground state. In Fig. 3b the soscaled IntCC/IntCN is shown for all phospholipids studied in the present work. Above ~125 K the IntCC/IntCN ratio deviates from its lowest-temperature value. In initial part of this deviation the behavior of the scaled ratio is similar for DOPC, POPC, PC24 and DPPC. The following sharper decrease of IntCC/IntCN is different for the different lipids and in the case of DOPC, POPC and DPPC is ended by a breaking step at Tm. In the case of PC24 the experimental IntCC/IntCN looks also step-like near Tm. Only in the case of DLPC the peculiarity near Tm is not pronounced. Figure 4 illustrates the representative Raman spectra in the spectral ranges of the C–H stretch vibrations for the PC24, DLPC and DOPC bilayers. The Raman spectra for DPPC and POPC bilayers were similar to those presented in refs. 26, 28. The prominent features of Raman spectra of the phospholipids are the lines near 2850 cm-1 and 2880 cm-1 attributed to symmetric and antisymmetric CH2 stretching vibrations, respectively. These lines are sharp in the gel phase of phospholipid vesicles, and the intensity of the symmetric CH2 stretching line is almost temperature independent. When temperature increases the Raman intensity of asymmetrical methylene stretching line decreases, this line is not manifested in a featureless background of other CH-stretching contribution in the fluid phase (Figure 4). This background impedes the decomposition of the Raman spectrum into separate contours.55 Instead of this the ratio of



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intensities for symmetrical IsCH and asymmetrical IaCH methylene stretching bands is considered. This ratio is believed to be sensitive to lateral intermolecular packing and interaction, also to the intrachain conformational disorder.52,56-59 Often this ratio is used to characterize the gel-to-fluid phase transition.16-18,60-63 The temperature dependence of the ratio R = IaCH/IsCH is shown in Figure 5a for all phospholipids studied. It is seen that R tends to a constant value in the low-temperature limit. The temperature raise decreases the value of R, which reaches its minimum in the fluid state. At the gel-fluid phase transition the parameter R obeys discontinuity in the case of PC24, DPPC, POPC and DOPC bilayers. In the case of DLPC a maximum of the derivative of RCH(T) with respect to temperature is observed near Tm. The absolute values of R in the low-temperature limit and in the fluid phase are different for different phospholipids (Figure 5a). To visualize peculiarities of the temperature evolution of RCH, the temperature dependence of sc (T ) = RCH

RCH (T ) − RCH ( fluid ) RCH (T = 0) − RCH ( fluid )

(2)

sc (T ) can be described as a temperature independent is shown in Figure 5b. Qualitatively, RCH

behavior at low temperatures, which is replaced by a noticeable decrease. Onset of this decrease is ~ 100 K for the bilayers of unsaturated lipids (POPC, DOPC, DLPC), ~ 180 K and ~ 230 K for the saturated lipid DPPC and PC24, respectively.

Discussion We see that the fraction of the hydrocarbon chains in the ground state decreases as temperature increases (Figure 3b). This decrease is caused by an occupation of newly achievable conformations of the lipid tails. Formally, a lot of different states can be considered. For example, in the framework of the Pink model64 there are 10 allowed conformational states of the chain, treated in a triangular lattice. The Pink model considers all-trans state of the chain conformation, three states with one gauche conformation, three states with two gauche conformations, two states with three gauche conformations, and high-energy “melted” state. Among the states with two gauche conformations a particular importance has the so-called “kink” conformation, which includes gauche+–trans–gauche– sequence and results in small penalty for lateral area of phospholipid molecule.64 The Pink model takes into account the energy of interaction between two neighbor chains and effective lateral pressure. This model can be analyzed only numerically, however, Figure 5 of the original work64 evidences that all-trans, kink and excited melted states dominate in the numerical solution for the gel phase. Recently it was shown that a model with only two excited states of hydrocarbon chains describes well the temperature dependence of RCC(T) of the DPPC and POPC bilayers.27,29 In this model one of the 8 ACS Paragon Plus Environment

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excited state is the kink state, and the second one is a highly disordered “melted” state. We will follow this approach, which is a fruitful compromise between the models, taking in account two conformational (ground and melted) states65,66 or ten lipid conformational states.63,67,68 However, we should take into account that if one hydrocarbon chain of a phospholipid molecule is in the melted state, it is expected that the second hydrocarbon chain of this molecule is also in the melted state (see for illustration figures in ref 69). The previous versions of the twoexcited-state27 or Pink64 models neglected the fact that we should consider the melted state as a state for the phospholipid molecule, not for a particular hydrocarbon chain. To rewrite formulas of the two-excited-state model we will consider the ground and melted state of the molecule, when both hydrocarbon chains are either in the ground or in the melted state (see Figure 6 for illustration). The melted state of the molecule is characterized by the degeneracy gm and the excitation energy Um. The values of the degeneracy are determined relative the degeneracy of the ground state. The kink state of a chain is considered to be independent of the state of the second chain of the molecule. Thus, we count the cases, when only one hydrocarbon chain is in the kink state, while the second is in the ground state, and when both chains are in the kink state. We will describe the kink state of a hydrocarbon chain by the degeneracy gk and by the excitation energy Uk. In this case the molecule state with one kink chain and one ground chain is characterized by the degeneracy 2gk and by the excitation energy Uk. The molecule state with two kink chains is characterized by the degeneracy g k2 and by the excitation energy 2Uk. In the ratio RCC(T) the contribution from the molecule with kink and ground chains is half of the contribution from the molecule with only ground chains. Summing two contributions (from the molecules in the ground state and from the molecules with only one chain in the ground state) and doing a simple conversion we get the expression,

RCC (T ) =

1 + g k exp(− U k / k B T ) . (1 + g k exp(− U k / k BT ))2 + g m exp( −U m / k BT )

(3)

Usually, in numerical simulations the values of degeneracy are calculated with the framework of a model used.64,70 Here, we will use eq 3 to fit the experimental data and to extract the values of degeneracy from the experiment, also as the values of the excitation energies. Note that eq 3 can be applied also for the case when the ground state has the degeneracy more than one. In this case gk and gm are the ratios between the degeneracy of an excited state and one of the ground state. It is expected that the values of Uk and Um can be significantly different. In this case a temperature range can exist where reducing the number of chains in the ground state is ruled by excitation to the kink state only. In this case RCC(T) is described by 9 ACS Paragon Plus Environment


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RCC (T ) =

1 . g k exp(− U k / k B T ) + 1

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(4)

If the kink degeneracy and excitation energy are the same for all phospholipids, then an universal description of RCC(T) is expected for the temperature range, where excitations to the kink state dominate. Indeed, this universal description by eq 4 with Uk/kB = 650 K, gk = 2.5 is seen in Figure 3b. The estimated energy of the kink state, Uk/kB = 650 K, corresponds well to the double value of the energy εg needed to form a single gauche bond (εg/kB ~ 325 K),64 as it would be expected. Thus, at enough low temperatures the thermal evolution of the conformational states of hydrocarbon chains is described by the excitation into the kink state. Behavior of RCC(T) proves this in the case of PC24, DPPC, POPC and DOPC, and the data for DLPC does not contradict this statement (Figure 3b). In the temperature range, where the excitations to the highly disordered melted states become important, the universality of eq 4 fails. The fit by eq 3, which includes the contribution from the melted states, was done for the experimental RCC(T) in the gel phase of the bilayers, from the lowest temperature to Tm. Here, values of the kink degeneracy and excitation energy were fixed to the values found from the fit by eq 4 (Uk/kB = 650 K and gk = 2.5). The fitting curves for the phospholipid bilayers are shown in Figure 3b. These fits work very well for DLPC, DOPC, POPC and DPPC. In case of PC24 eq 3 describes the experimental data reasonably well, but probably the approximation of one melted state is too coarse for this phospholipid. Indeed, the long-chain diacylphosphatidylcholines are known by its complex phase behavior in the gel phase near Tm.8 Nevertheless, to avoid ambiguities in the fitting parameters (if eq 3 would be extended to the case of two different melted states), in the present work we have restricted ourselves to the case of eq 3 with one melted state. The parameters of the fits by eq 3 are presented in Table for all phospholipids. For convenience the logarithm of gm is presented in Table 1, a = log g m . Note that eq 3 of the present version of the two-excited-state model has the same capability in description of the experimental Raman data as the previous version.27 The difference between the versions is whether gm and Um should be attributed to the whole phospholipid molecule or to one chain only. This is important for the comparison of Um with the enthalpy of the main phospholipid transition and for the estimation of the number of gauche rotamers. It is usually believed that the changes in the chain conformational states contribute significantly to the enthalpy of the main phospholipid transition.1 In the framework of the twoexcited-state model the fluid phase corresponds to the hydrocarbon chains in the melted state, while they are mainly in the ground state for the gel state. Thus, it is expected that the part of the 10 ACS Paragon Plus Environment

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enthalpy related to the conformation changes during the gel-fluid transition should be equal to the energy difference between the ground and melted state. In this case the value of Um should be the same order as ∆H. Indeed Table 1 shows that values of Um and ∆H have the same order of magnitude (1 kcal/mol corresponds to 503.3 K). This agreement supports the assumption that the melted state of phospholipids molecules corresponds to highly disordered states, which are inherent for the fluid phase. Figure 7a inspects the possible correlation between Um and ∆H. It is seen that these two parameters are interrelated, supporting the description of the Raman data by the two-excitedstate model. Quantitative agreement is not excellent. There are few reasons for this. The main one is the excessive simplicity of the model, which neglects by the collective character of transition, the distribution of melted states, lateral ordering/disordering processes, and interaction between the two monolayers and so on. The enthalpy of the phase transition has additional contributions out of scope of the two-excited-state model, which shifts the predicted ∆H to higher values than it is shown in Figure 7a by the line. On the other hand, according Figure 3b, a part of the hydrocarbon chains is already in the melted state near Tm. Using eq 3 we can predict the part of the hydrocarbon chains in the melted state, nm, as function of RCC(T) via n m (T ) = 1 − RCC (T ) ⋅ (1 + g k exp(− U k / k B T )) ,

(5)

which is valid also in the case of a distribution of gm and Um. The experimental RCC(T) and eq 5 provide estimations of nm(T→Tm) = 0.23, 0.27, 0.21, 0.33, and 0.45 for DLPC, DOPC, POPC, DPPC, and PC24, respectively. Correspondingly, the conformational contribution to ∆H can be less by these parts. This shifts the predicted ∆H to lower values than it is expected in Figure 7a. Thus, there are factors which can shift the predicted ∆H(Um) to higher or to lower values that explains the scatter of the points around the line in Figure 7a. In spite of these factors there is the qualitative correlation between Um and ∆H in Figure 7a. This agreement supports the description of the melted state of phospholipids as the states, which are inherent for the fluid phase. Let’s consider a simple model of the melted state of chains and characterize the conformational state by a number of carbon bond N, which are released to take one of three orientations (in spirit of ref 64). These orientations form trans or gauche conformations, separated by the energy εg, which is difference between energies of gauche and trans conformations of bonds. In the ground state of chains these freedom degrees are frozen. Then the melt state degeneracy gm = 3N and a = log g m = N log 3

(6)

is expected. Within the model the number of gauche rotamers is 2N/3, and the energy related to excitation of gauche rotamers is 2ε g N / 3 . Also, the energy of the melted state includes a work 11 ACS Paragon Plus Environment


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fA, which is paid for a change of lateral lipid area in transition from the ground state to the melted state:

U m = 2ε g N / 3 + f A .

(7)

Comparison of eq 6 and eq 7 predicts a correlation between a and Um,

a=

3 log 3 (U m − f A ) . εg 2

(8)

As the first approximation eq 8 can be tested in neglecting fA. Figure 7b shows the interrelation between the fitting parameters of the Raman data (Table 1). The expectation from eq 8 with the known value of εg/kB = 325 K and fA = 0 is in reasonable agreement with the experimental points (Figure 7b). Thus, the simple model of the melted state of chains predicts the right order of magnitude for a and Um. Agreement in Figure 7b can be improved by taking into account fA. For example, if f A = ε A N , which assumes that the lipid lateral radius increases as

N , then a linear

correlation

a=

log 3 ⋅ U m 2ε g / 3 + ε A

(9)

is expected. The fit in Figure 7b provides the estimation of ε A / k B ≈ 100 K . From eq 6 and experimental value of a the estimation of N can be done. The estimations of N are 3.3 ± 0.6, 12.4 ± 1.5, 13.6 ± 0.6, 17.0 ± 3.6, and 17.4 ± 5.0 for DLPC, DOPC, POPC, DPPC, and PC24, respectively. This is an effective number of C-C bonds taking part in transgauche switching in the melted state (for two hydrocarbon tails of a molecule). It is seen that for saturated lipids the estimated N is about half of the carbon number in hydrocarbon tails. For lipids with monounsaturated tails (POPC and DOPC) it is about one third of the carbon number in hydrocarbon tails. In the case of DLPC N is several times smaller than the carbon number in hydrocarbon tails. Thus we can conclude that the transformation from the ground state to the melted state needs less conformation changes in the case of unsaturated phospholipids in comparison with the saturated ones. Our estimation of N in the case of DPPC means about 5.7 ± 1.2 gauche rotamers per chain in the fluid phase. Experimental techniques based on the analysis of spectra of CH2 groups (IR absorption spectroscopy71,72 and NMR spectroscopy73) and computer simulations74,75 provide somehow lower estimations of the number of gauche rotamers in the fluid phase of DPPC, which are about 3.5 – 4.4 per chain. Nevertheless, the agreement between the experimental data and our very simple microscopic description of the two-excited-state model should be considered as reasonably good.


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Analysis of RCH(T) found from the Raman intensities of symmetric and antisymmetric CH2 stretching vibration lines (Figure 5) reveals that this ratio is sensitive not only to conformational states, but also to other degrees of freedom of the phospholipids. This agrees with usual belief about the sensitivity of antisymmetric CH2 stretching vibrations to lateral intermolecular packing and interaction.52,56-59 Similar conclusions were done in our previous works28,29 from the comparison of RCH(T) behavior in DPPC and POPC. It is interesting to see (Figure 5b) that the onset of decrease in RCH(T) (~ 100 K) is the same for all unsaturated phospholipids. It can be attributed to effect of packing defects, which are inherent for unsaturated phospholipids. These defects can release the freedom degrees, which are responsible for reducing RCH(T). In particular the temperature of the RCH(T) onset can be ascribe to release the skeletal torsions,59 when the packing defects (e.g. free-volume like holes29) are present. Another interesting observation is the behavior of RCH(T) in the gel phase near Tm. An unexpected decrease of the slope of RCH(T) is seen in this temperature range in the case of the saturated lipids (Figure 5b), which is in contrast to the RCC(T) behavior (Figure 3b). This means that near Tm the increase of the melted states of saturated hydrocarbon chains does not release new degrees of freedom, which are responsible for RCH(T). It is remarkable that while RCC(T) and RCH(T) measure the different degrees of freedom, they are very similar for the POPC and DOPC bilayers (Figures 3 and 5). This result has the natural explanation that at low temperatures the saturated hydrocarbon chain of POPC takes the same conformation as the monounsaturated chain. Thus the POPC and DOPC tail conformations are similar in the gel phase.

Conclusions The Raman spectra of bilayers of five phosphatidylcholines (DLPC, DOPC, POPC, DPPC and PC24) were studied in a wide temperature range. Raman line intensities of CH2stretching modes are sensitive to the temperature evolution of lateral interchain packing and interactions. It was found that the onset temperature of the decrease of the IaCH/IsCH ratio is the same for the unsaturated lipids (DLPC, POPC and DOPC), that was interpreted as effect of the packing defects in the bilayers of these phospholipids. The chain conformational order was characterized by the intensity of the high-frequency C-C stretching modes ICC. An approach was used where the Raman intensity of this mode serves as a measure of the hydrocarbon chains in the ground conformational state. It was shown that even in the gel phase the occupation of the ground state decreases as the temperature increases and the excitation to higher-energy conformational states occurs.



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The modified two-excited-state model describes well the temperature dependences of the ICC/ICN ratio. At temperatures significantly below Tm the deviation of the ICC/ICN ratio from the low-temperature limit is well described by excitations to the kink state. The values of the excitation energy (about of two energies for forming a gauche bond) and of the degeneracy were the same for all lipids studied. The excitation energy and the degeneracy of highly disordered (melted) state are different for different phospholipids. These energies have the same order of magnitude as the enthalpy of the gel-fluid transition. The degeneracy of the melted conformation state correlates with the value of the excitation energy. This relation can be interpreted by a simple microscopic model, which provides an estimation of the effective number of C-C bonds taking part in trans-gauche switching in the melted state. Thus, we conclude that the experimental ICC/ICN ratio and the two-excited-state model describe the temperature evolution of the conformational state of hydrocarbon chains in the gel phase. A particular outcome of our study is the statement about the similarity of the lateral and conformational packing for DOPC and POPC in the gel state. This statement was done from the similarity of the IaCH/IsCH and ICC/ICN ratios for these phospholipids.

Acknowledgements Authors would like to acknowledge S.V. Adichtchev and A.M. Pugachev for the help in the DSC experiment.


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References (1) Nagle, J. F. Theory of the Main Lipid Bilayer Phase Transition. Ann. Rev. Phys. Chem. 1980, 31, 157-195. (2) Binder, H.; Gutberlet, T.; Anikin. A.; Klose, G. Hydration of the Dienic Lipid Dioctadecadienoylphosphatidylcholine in the Lamellar Phase – An Infrared Linear Dichroism and X-ray Study on Headgroup Orientation, Water Ordering, and Bilayer Dimension. Biophys. J. 1998, 74, 1908-1923. (3) Nagle, J. F.; Tristram-Nagle, S. Structure of Lipid Bilayers. Biochim. Biophys. Acta 2000, 1469, 159-195. (4) Naumann, C.; Brumm, T.; Bayerl, T. M. Phase Transition Behavior of Single Phosphatidylcholine Bilayers on a Solid Spherical Support Studied by DSC, NMR and FT-IR. Biophys. J. 1992, 63, 1314-1319. (5) Schom, K.; Marsh, D. Dynamic Chain Conformation in Dimyristoyl Glycerol-Dimyristoyl Phosphatidylcholine Mixture. 2H-NMR Studies. Biophys. J. 1996, 71, 3320-3329. (6) Heimburg, T.; Würz, U.; Marsh, D. Binary Phase Diagram of Hydrated DimyristoylglycerolDimyristoylphosphatidylcholine Mixtures. Biophys. J. 1992, 63, 1369-1378. (7) Casal, H. L.; Mantsch, H. H. Polymorphic Phase Behavior of Phospholipid Membranes Studied by Infrared Spectroscopy. Biochim. Biophys. Acta 1984, 779, 381-401. (8) Snyder, R. G.; Liang, G. L.; Strauss, H. L.; Mendelsohn, R. IR Spectroscopic Study of the Structure and Phase Behavior of Long-Chain Diacylphosphatidylcholines in the Gel State. Biophys. J. 1996, 71, 3186-3198. (9) Villalaín, J. Membranotropic Effects of Arbidol, a Broad Anti-viral Molecule, on Phospholipids Model Membranes. J. Phys. Chem. B 2010, 114, 8544-8554. (10) Schultz, Z. D.; Levin, I. W. Vibrational Spectroscopy of Biomembranes. Annu. Rev. Anal. Chem. 2011, 4, 343-366. (11) Pfeiffer, H.; Klose, G.; Heremans, K. FTIR Spectroscopy Study of the Pressure-Dependent Behaviour of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-Palmitoyl-2oleolyl-sn-glycero-3-phosphocholine (POPC) at Low Degrees of Hydration. Chem. Phys. Lipids 2013, 170-171, 33-40. (12) Marsh, D.; Watts, A.; Knowles, P. F. Evidence for Phase Boundary Lipids. Permeability of Tempo-choline into Dimyristoylphosphatidylcholine Vesicles at the Phase Transition. Biochemistry 1976, 15, 3570-3578. (13) Moser, M.; Marsh, D.; Meier, P.; Wassmer K.-H.; Kothe, G. Chain Configuration and Flexibility Gradient in Phospholipids Membranes. Comparison between Spin-label



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 37

Electron Spin Resonance and Deuteron Nuclear Magnetic Resonance, and Identification of New Conformatons. Biophys. J. 1989, 55, 111-123. (14) Reboiras, M. D.; Marsh, D. EPR Studies on the Influence of Chainlength on the Segmental Motion of Spin-labelled Fatty Acids in Dimyristoylphosphatidylcholine Bilayers. Biochim. Biophys. Acta 1991, 1063, 259-264. (15) Watts, A.; Masrch, D.; Knowles, P. F. Characterization of Dimyristoylphosphatidylcholine Vesicles and their Dimensional Changes through the Phase Transition: Molecular Control of Membrane Morphology. Biochemistry 1978, 17, 1792-1801. (16) Lefevre, T.; Picquart, M. Vitamin E-phospholipid Interaction in Model Multilayer Membranes: A Spectroscopic Study. Biospectroscopy 1996, 2, 391-403. (17) Procházka, M.; Štěpánek, J.; Turpin, P.-Y. Interaction of Phospholipids Dispersion with Water-Soluble Porphyrins as Monitored by their Raman Temperature Profiles. Chem. Phys. Lipids 2004, 132, 145-156. (18) Fox, C. B.; Uibel, R. H.; Harris, J. M. Detecting Phase Transitions in Phosphatidylcholine Vesicles by Raman Microscopy Self-Modeling Curve Resolution. J. Phys. Chem. B 2007, 111, 11428-11436. (19) Sassi, P.; Caponi, S.; Ricci, M.; Morresi, A.; Oldenhof, H.; Wolkers, W.F.; Fioretto, D. Infrared Versus Light Scattering Techniques to Monitor the Gel to Liquid Crystal Phase Transition in Lipid Membranes. J. Raman Spectrosc. 2015, 46, 644-651. (20) Kaasgaard, T.; Mouritsen, O. G.; Jørgensen, K. Freeze/Thaw Effects on Lipid-Bilayer Vesicles Investigated by Differential Scanning Calorimetry. Biochim. Biophys. Acta

2003, 1615, 77-83. (21) Corvera, E.; Laradji, M.; Zuckermann, M. J. Application of Finite-Size Scaling to the Pink Model for Lipid Bilayers. Phys. Rev. E 1993, 47, 696-703. (22) Egberts, E.; Marrink, S.-J.; Berendsen, H. J. C. Molecular Dynamics Simulation of a Phospholipids Membrane. Eur. Biophys. J. 1994, 22, 423-436. (23) Tristram-Nagle, S.; Nagle, J.F. Lipid Bilayers: Thermodynamics, Structure, Fluctuations, and Interactions. Chem. Phys. Lipids 2004, 127, 3-14. (24) Fitter, J.; Lechner, R. E.; Dencher, N. A. Interaction of Hydration Water and Biological Membranes Studied by Neutron Scattering. J. Phys. Chem. B 1999, 103, 8036-8050. (25) Bartucci, R.; Erilov, D. A.; Guzzi, R.; Sportelli, L.; Dzuba, S. A.; Marsh, D. Time-Resolved Electron Spin Resonance Studies of Spin-Labelled Lipids in Membranes. Chem. Phys. Lipids 2006, 141, 142-157.


Page 17 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(26) Surovtsev, N. V.; Salnikov, E. S.; Malinovsky, V. K.; Sveshnikova, L. L.; Dzuba, S. A. On the Low-Temperature Onset of Molecular Flexibility in Lipid Bilayers Seen by Raman Scattering. J. Phys. Chem. B 2008, 112, 12361-12365. (27) Surovtsev, N. V.; Dzuba, S. A. Conformational Changes of Lipids in Bilayers at the Dynamical Transition Near 200 K Seen by Raman Scattering. J. Phys. Chem. B 2009, 113, 15558-15562. (28) Surovtsev, N. V.; Dzuba, S. A. Flexibility of Phospholipids with Saturated and Unsaturated Chains Studied by Raman Scattering: The Effect of Cholesterol on Dynamical and Phase Transitions. J. Chem. Phys. 2014, 140, 235103. (29) Surovtsev, N. V.; Ivanisenko, N. V.; Kirillov, K. Yu.; Dzuba, S. A. Low-Temperature Dynamical and Structural Properties of Saturated and Monounsaturated Phospholipid Bilayers Revealed by Raman and Spin-Label EPR Spectroscopy. J. Phys. Chem. B 2012, 116, 8139-8144. (30) http://www.avantilipids.com. (31) Svetlovics, J. A.; Wheaten, S. A.; Almeida, P. F. Phase Separation and Fluctuations in Mixtures of a Saturated and an Unsaturated Phospholipid. Biophys. J. 2012, 102, 25262535. (32) Tenchov, B. G.; Yao, H.; Hatta, I. Time-Resolved X-ray Diffraction and Calorimetric Studies at Low Scan Rates. I. Fully Hydrated Dipalmitoylphosphatidylcholine (DPPC) and DPPC/Water/Ethanol Phases. Biophys. J. 1989, 56, 757-768. (33) Mabrey, S.; Sturtevant, J. M. Investigation of Phase Transition of Lipids and Lipid Mixture by High Sensitivity Differential Scanning Calorimetry. Proc. Natl. Acad. Sci. USA 1976, 73, 3862-3866. (34) Ebel, H; Grabitz, P.; Heimburg, T. Enthalpy and Volume Changes in Lipid Membranes. I. The Proportionality of Heat and Volume Changes in the Lipid Melting Transition and Its Implication for the Elastic Constants. J. Phys. Chem. B 2001, 105, 7353-7360. (35) Fujisawa , S.; Kadoma, Y.; Masuhara, E. A Calorimetric Study of the Interaction of Synthetic Phospholipids Liposomes with Lipid-Soluble Small Molecules Used as Dental Materials and Devices. J. Biomed. Mater. Res. 1987, 21, 89-98. (36) Chowdhry, B. Z.; Lipka, G.; Sturtevant, J.M. Thermodynamics of Phospholipids-Sucrose Interaction. Biophys. J. 1984, 46, 419-422. (37) Bonora, S.; Toreggiani, A.; Fini, G. DSC and Raman study on the Interaction Between Polychlorinated Biphenyls (PCB) and Phospholipids Liposomes. Thermochim. Acta

2003, 408, 55-65. 17 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 37

(38) Blume, A. Apparent Molar Heat Capacities of Phospholipids in Aqueous Dispersion. Effects of Chain Length and Head Group Structure. Biochemistry 1983, 22, 5436-5442. (39) Schwarz, F. P. Biological Thermodynamic Data for the Calibration of Differential Scanning Calorimeters: Dynamic Temperature Data on the Gel to Liquid Crystal Phase Transition of Dialkylphosphatidylcholine in Water Suspensions. Thermochim. Acta 1991, 177, 285303. (40) Pfeiffer, H.; Klose, G.; Heremans, K. Reorientation of Hydration Water During the Thermotropic

Main

Phase

Transition

of

1-Palmitoyl-2-oleolyl-sn-glycero-3-

phosphocholine (POPC) Bilayers at Low Degrees of Hydration. Chem. Phys. Lett. 2013, 572, 120-124. (41) Curatolo, W.; Sears, B.; Neuringer, L.J. A Calorimetry and Deuterium NMR Study of Mixed Model Membranes of 1-Palmitoyl-2-oleylphosphatidylcholine and Saturated Phosphatidylcholines. Biochim. Biophys. Acta 1985, 817, 261-270. (42) Rappolt, M.; Vidal, M. F.; Kriechbaum, M.; Steinhart, M.; Amenitsch, H.; Bernstorff, S.; Laggner, P. Structural, Dynamic and Mechanical Properties of POPC at Low Cholesterol Concentration Studied in Pressure/Temperature Space. Eur. Biophys. J. 2003, 31, 575585. (43) Ichimori, H.; Hata, T.; Matsuki, H.; Kaneshina, S. Effect of Unsaturated Acyl Chains on the Thermotropic and Barotropic Transitions of Phospholipid Bilayer Membranes. Chem. Phys. Lipids 1999, 100, 151-164. (44) Davis, P. J.; Fleming, B. D.; Coolbear, K. P.; Keough, K. M. W. Gel to Liquid-Crystalline Transition Temperatures of Water Dispersions of Two Pairs of Positional Isomers of Unsaturated Mixed-Acid Phosphatidylcholines. Biochemistry 1981, 20, 3633-3636. (45) Tada, K.; Miyazaki, E.; Goto, M.; Tamai, N.; Matsuki, H.; Kaneshina, S. Barotropic and Thermotropic Bilayer Phase Behavior of Positional Isomers of Unsaturated Mixed-Chain Phosphatidylcholines. Biochim. Biophys. Acta 2009, 1788, 1056-1063. (46) Rodríguez, G.; Rubio, L.; Barba, C.; López-Iglesias, C.; de la Maza, A.; López, O.; Cócera, M. Characterization of New DOPC/DHPC Platform for Dermal Applications. Eur. Biophys. J. 2013, 42, 333-345. (47) Davis, P. J.; Keough, K. M. W. Differential Scanning Calorimetric Studies of Aqueous Dispersions

of

Mixtures

of

Cholesterol

with

Mixed-Acid

and

Single-Acid

Phosphatidylcholines. Biochemistry 1983, 22, 6334-6340. (48) Fa, N.; Ronkart, S.; Schanck, A.; Deleu, M.; Gaigneaux, A.; Goormaghtigh, E.; MingeotLeclercq, M.-P. Effect of the Antibiotic Azithromycin on Thermotropic Behavior of DOPC or DPPC Bilayers. Chem. Phys. Lipids 2006, 144, 108-116. 18 ACS Paragon Plus Environment

Page 19 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(49) Findlay, F. J.; Barton, P. G. Phase Behavior of Synthetic Phosphatidylglycerol and Binary Mixture with Phosphatidylcholines in the Presence and Absence of Calcium Ions. Biochemistry 1978, 17, 2400-2405. (50) Fritzsching, K. J.; Kim, J.; Holland, G. P. Probing Lipid-Cholesterol Interactions in DOPC/eSM/Chol and DOPC/DPPC/Chol Model Lipid Raft with DSC and

13

C Solid-

State NMR. Biochim. Biophys. Acta 2013, 1828, 1889-1898. (51) Kariel, N.; Davidson, E.; Keough K.M.W. Cholesterol Does not Remove the Gel-Liquid Crystalline Phase Transition of Phosphatidylcholines Containing Two Polyenoic Acyl Chains. Biochim. Biophys. Acta 1991, 1062, 70-76. (52) Gaber, B. P.; Peticolas, W. L. On the Quantitative Interpretation of Biomembrane Structure by Raman Spectroscopy. Biochim. Biophys. Acta 1977, 465, 260-274. (53) Meier, R. J. Studying the Length of Trans Conformational Sequences in Polyethylene Using Raman Spectroscopy: A Computational Study. Polymer 2002, 43, 517-522. (54) McCreery, R. L. Raman Spectroscopy for Chemical Analysis; John Wiley & Sons: New York, USA, 2000. (55) Harrand, M. Polarized Raman Spectra of Oriented Dipalmitoylphosphatidylcholine (DPPC). I. Scattering Activities of Skeletal Stretching and Methylene Vibrations of Hydrocarbon Chains. J. Chem. Phys. 1983, 79, 5639-5651. (56) Yellin, N.; Levin, I. W. Hydrocarbon Chain Disorder in Lipid Bilayers. Temperature Dependent Raman Spectra of 1,2-Diacyl Phosphatidylcholine-Water Gels. Biochim. Biophys. Acta 1977, 489, 177-190. (57) Levin, I. W.; Thompson, T. E.; Barenholz, Y.; Huang, C. Two Types of Hydrocarbon Chain Interdigitation in Sphingomyelin Bilayers. Biochemistry 1985, 24, 6282-6286. (58) Wallach, D. F. H.; Verma, S. P.; Fookson, J. Application of Laser Raman and Infrared Spectroscopy to the Analysis of Membrane Structure. Biochim. Biophys. Acta 1979, 559, 153-208. (59) Zerbi, G.; Roncone, P.; Longhi, G.; Wunder, S. L. Molecular Flexibility of Polymethylene Molecules: A Raman Spectroscopic Study. J. Chem. Phys. 1988, 89, 166-173. (60) Bunow, M. R.; Levin, I. W. Vibrational Raman Spectra of Lipid Systems Containing Amphotericin B. Biochim. Biophys. Acta 1977, 464, 202-216. (61) Verma, S. P.; Wallach, D. F. H. Multiple Thermotropic State Transition in Erythrocyte Membranes. A Laser-Raman Study of the CH-Stretching and Acoustical Regions. Biochim. Biophys. Acta 1976, 436, 307-318. (62) Weidkamm, E.; Bamberg, E.; Brdiczka, D.; Wildermuth, G.; Macco, F.; Lehmann, W.; Weber, R. Raman Spectroscopic Investigation of the Interaction of Gramicidin A With 19 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 37

Dipalmitoyl Phosphatidylcholine Liposomes. Biochim. Biophys. Acta 1977, 464, 442447. (63) Wong, P. T. T.; Mantsch, H. H. Effects of Hydrostatic Pressure on the Molecular Structure and Endothermic Phase Transitions of Phosphatidylcholine Bilayers: A Raman Scattering Study. Biochemistry 1985, 24, 4091-4096. (64) Pink, D. A.; Green, T. J.; Chapman, D. Raman Scattering in Bilayers of Saturated Phosphatidylcholines. Experiment and Theory. Biochemistry 1980, 19, 349-356. (65) Heimburg, T. A; Biltonen R. L. A Monte Carlo Simulation Study of Protein-Induced Heat Capacity Changes and Lipi-Induced Protein Clustering. Biophys. J. 1996, 70, 84-96. (66) Jerala, R.; Almeida, P. F. F.; Biltonen, R. L. Simulation of the Gel-Fluid Transition in a Membrane Composed of Lipids with Two Connected Acyl Chains: Application of a Dimer-Move Step. Biophys. J. 1996, 71, 609-615. (67) Zhang, Z.; Laradji, M.; Guo, H.; Mouritsen, O. G.; Zuckermann, M. J. Phase Behavior of Pure Lipid Bilayers with Mismatch Interaction. Phys. Rev. A 1992, 45, 7560-7567. (68) Sadeeghu, S.; Vink, R. L. C. Main Transition in the Pink Membrane Model: Finite-Size Scaling and the Influence of Surface Roughness. Phys.Rev. E 2012, 85, 61912. (69) Heimburg, T. A Model for the Lipid Pretransition: Coupling of Ripple Formation with the Chain-Melting Transition. Biophys. J. 2000, 78, 1154-1165. (70) Mouritsen, O. G.; Boothroyd, A.; Harris, R.; Jan, N.; Lookman, T.; MacDonald, L.; Pink, D. A.; Zuckermann, M. J. Compute Simulation of the main Gel-Fluid Phase Transition of Lipid Bilayers. J. Chem. Phys. 1983, 79, 2027-2041. (71) Casal, H. L.; McElhaney, R. N. Quantitative Determination of Hydrocarbon Chain Conformation Order in Bilayers of Saturated Phosphatidylcholines of Various Chain Lengths by Fourier Transform Infrared Spectroscopy. Biochemistry 1990, 29, 5423-5427. (72) Mendelsohn, R.; Davies, M. A.; Brauner, J. W.; Schuster, H.F.; Dluhy, R. A. Quantitative Determination of Conformational Disorder in the Acyl Chains of Phospholipid Bilayers by Infrared Spectroscopy. Biochemistry 1989, 28, 8934-8939. (73) Douliez, J.-P.; Léonard, A.; Dufourc, E. J. Restatement of Order Parameters in Biomembranes: Calculation of C-C Bond Order Parameters from C-D Quadrupolar Splittings. Biophys. J. 1995, 68, 1727-1739. (74) Jämbeck, J. P. M.; Lyubartsev, A. P. Derivation and Systematic Validation of a Refined AllAtom Force Field for Phosphatidylcholine Lipids. J. Phys. Chem. B 2012, 116, 31643179.


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(75) Leekumjorn, S. Sum, A. K. Molecular Studies of the Gel to Liquid-Crystalline Phase Transition for Fully Hydrated DPPC and DPPE Bilayers. Biochim. Biophys. Acta 2007, 1768, 354-365.



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Table 1. Gel-fluid transition parameters of various phospholipids and the fit parameters of the Raman data by Eq. (3). Lipid

Tm (K)

∆H (kcal/mol)

Um/kB (K)

a = log g m

DLPC

216 ± 1

1.1 ± 0.3

1010 ± 150

1.6 ± 0.3

DOPC

255.5 ± 0.5

8.2 ± 0.6

3580 ± 450

5.9 ± 0.7

POPC

271 ± 1

4.6 ± 0.8

4280 ± 200

6.5 ± 0.3

DPPC

315.5 ± 0.5

8.1 ± 0.5

5810 ± 1250

8.1 ± 1.7

PC24

352.5 ± 1

17 ± 1.0

6360 ± 1900

8.3 ± 2.4


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Figure captions Figure 1. Chemical formulas of phospholipids used in the study. The main transition temperatures Tm are shown according to ref 30.

Figure 2. Representative Raman spectra of PC24 (a), DLPC (b), and DOPC (c) bilayers in the CN and C-C stretching spectral ranges. Spectra are normalized to the same 720 cm-1 line intensity and shifted upwards for convenience.

Figure 3. a) The ratio of the integral Raman intensities of the C-C (~1130 cm-1) and C-N (730 cm-1) stretching lines, IntCC/IntCN, for PC24 (circles), DPPC (diamonds), POPC (down triangles), DOPC (squares), and DLPC (up triangles) versus temperature. b) The scaled ratio of IntCC/IntCN versus temperature (the same notation as in the top panel). Thin lines are fits by eq 3 from the lowest temperature to Tm. and thick line is eq 4.

Figure 4. Representative Raman spectra of PC24 (a), DLPC (b), and DOPC (c) bilayers in the CH stretching spectral range. Spectra are shifted upwards for convenience.

Figure 5. a) The ratio of intensities of the asymmetric (2880 cm-1) and symmetric (2850 cm-1) CH2 stretching lines, IaCH/IsCH, for PC24 (circles), DPPC (diamonds), POPC (down triangles), DOPC (squares), and DLPC (up triangles) bilayers versus temperature. b) The ratio R = IaCH/IsCH scaled according eq 2 versus temperature (the same notation as in the top panel).

Figure 6. Illustration of the ground, kink and melted states of hydrocarbon chains of phospholipids in the framework of the model.

Figure 7. a) ∆H/kB from the DSC experiment versus Um/kB, the fitting parameter in eq 3. The line is ∆H/kB = Um/kB. b) Fitting parameter a = log g m versus Um/kB. The solid line is eq 8 with f A = 0 and the dash line is the fit by eq 9.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

16:0 PC (DPPC), Tm = 314 K

16:0-18:1 PC (POPC), Tm = 271 K

18:1 PC (DOPC), Tm = 256 K

18:2 (Cis) PC (DLPC), Tm = 216 K

24:0 PC (PC24), Tm = 353 K

Fig. 1


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a)

PC24 50 K 150 K 250 K 330 K

Raman intensity [arb. un.]

Page 25 of 37

365 K

b)

DLPC 50 K 120 K 180 K 230 K 280 K

c)

DOPC 60 K 120 K 180 K 240 K 280 K

700

750

1050

1100

1150 -1

Raman shift [cm ] Fig. 2



3.0

a)

IntCC/IntCN

2.5 2.0 1.5 1.0 0.5 0.0 1.0

IntCC/IntCN scaled

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 37

b)

0.8 0.6 0.4 0.2 0.0

0

50 100 150 200 250 300 350 Temperature [K]

Fig. 3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PC24

a)

50 K 150 K 250 K 333 K


Page 27 of 37

363 K

b)

DLPC 50 K 120 K 180 K 230 K 280 K

c)

DOPC 70 K 140 K 210 K 265 K 310 K

2800

2850

2900

2950 -1



3000


2.4

Ratio IaCH/IsCH

2.2

a)

2.0 1.8 1.6 1.4 1.2 1.0 0.8 1.0

Scaled ratio IaCH/IsCH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 37

b) 0.8 0.6 0.4 0.2 0.0 0

50 100 150 200 250 300 350 Temperature [K]

Fig. 5

Fig. 6 28 ACS Paragon Plus Environment

PC24 8000 6000 DOPC

DPPC

4000 2000

LOG of degeneracy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Entalpy of transition [K]

Page 29 of 37

POPC DLPC

0 12

a)

DPPC

10 8

PC24

6

POPC DOPC

4 2

b)

DLPC 0 0

2000

4000

6000

8000

Apparent barrier of melted state [K] Fig. 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC image


Page 30 of 37

Page 31 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


16:0 PC (DPPC), Tm = 314 K

16:0-18:1 PC (POPC), Tm = 271 K

18:1 PC (DOPC), Tm = 256 K

18:2 (Cis) PC (DLPC), Tm = 216 K

24:0 PC (PC24), Tm = 353 K

Fig. 1

ACS Paragon Plus Environment


a)

PC24 50 K 150 K 250 K 330 K


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

Page 32 of 37

365 K

b)

DLPC 50 K 120 K 180 K 230 K 280 K

c)

DOPC 60 K 120 K 180 K 240 K 280 K

700

750

1050

1100

1150 -1

Raman shift [cm ] ACS Paragon Plus Environment

Fig. 2

Page 33 of 37

3.0

a)

IntCC/IntCN

2.5 2.0 1.5 1.0 0.5 0.0 1.0

IntCC/IntCN scaled

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59


b)

0.8 0.6 0.4 0.2 0.0

0

Fig. 3

50 100 150 200 250 300 350 Temperature [K] ACS Paragon Plus Environment


PC24

a)

50 K 150 K 250 K 333 K


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

Page 34 of 37

363 K

b)

DLPC 50 K 120 K 180 K 230 K 280 K

c)

DOPC 70 K 140 K 210 K 265 K 310 K

2800

2850

2900

2950 -1



3000

Page 35 of 37

2.4

Ratio IaCH /IsCH

2.2

a)

2.0 1.8 1.6 1.4 1.2 1.0 0.8 1.0

Scaled ratio IaCH /IsCH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59


b)

0.8 0.6 0.4 0.2 0.0 0

50 100 150 200 250 300 350 Temperature [K]

Fig. 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Illustration of the ground, kink and melted states of hydrocarbon chains of phospholipids in the framework of the model. 177x111mm (120 x 120 DPI)


Page 36 of 37


PC24 8000 6000 DOPC

DPPC

4000 2000

LOG of degeneracy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

Entalpy of transition [K]

Page 37 of 37

POPC DLPC

0 12

a)

DPPC

10 8

PC24

6

POPC DOPC

4 2

b)

DLPC 0 0

2000

4000

6000

8000

Apparent barrier of melted state [K] Fig. 7


Temperature-Dependent Hydrocarbon Chain Disorder in

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