Low-Temperature Dynamics of Chain-Labeled Lipids in Ester- and

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Low-Temperature Dynamics of Chain-Labelled Lipids in Ester- and Ether-Linked Phosphatidylcholine Membranes Erika Aloi, Maria Oranges, Rita Guzzi, and Rosa Bartucci J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b07386 • Publication Date (Web): 11 Sep 2017 Downloaded from http://pubs.acs.org on September 15, 2017

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Low-temperature

The Journal of Physical Chemistry

Dynamics

of

Chain-labelled

Lipids

in

Ester-

and

Ether-linked

Phosphatidylcholine Membranes

Erika Aloi,† Maria Oranges,†, ‡ Rita Guzzi,† Rosa Bartucci†,*

†

Department of Physics, Molecular Biophysics Laboratory, University of Calabria, 87036 Rende (CS),

Italy ‡

present address: School of Chemistry, University of St Andrews, St Andrews KY16 9ST Scotland,

UK

*corresponding author: [email protected]

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ABSTRACT

Continuous wave electron paramagnetic resonance spectroscopy and two-pulse echo detected spectra of chain-labelled lipids are used to study the dynamics of frozen lipid membranes over the temperature range 77-260 K. Bilayers of ester-linked dihexadecanoyl phosphatidylcholine (DPPC) with noninterdigitated chains and ether-linked dihexadecyl phosphatidylcholine (DHPC) with interdigitated chains are considered. Rapid stochastic librations of small angular amplitude are found in both lipid matrices. In noninterdigitated DPPC bilayers, the mean-square angular amplitude, 〈 〉, of the motion increases with temperature and it is larger close to the chain termini than close to the polar/apolar interface. In contrast, in interdigitated DHPC lamellae 〈 〉 is small and temperature and label-position independent at low temperature and increases steeply at high temperature. The rotational correlation time, , of librations lies in the subnanosecond range for DPPC and in the nanosecond range for DHPC. In all membrane samples, the temperature dependence of 〈 〉 resembles that of the meansquare atomic displacement revealed by neutron scattering and a dynamical transition is detected in the range 210-240 K. The results highlight the librational oscillations and the glass-like behaviour in bilayer and interdigitated lipid membranes.


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INTRODUCTION

Several biophysical studies of macromolecules and supramolecular assemblies are performed at low, cryogenic temperature by using high resolution spectroscopic techniques sampling different timescales.1-4 Low-temperature allows one to unravel specific kinetic, structural and dynamic features that are present also at higher physiological temperatures. The studies therefore play an important role in elucidating the molecular mechanisms underlying structure/function relations in biosystems. Moreover, biological media are intensively investigated at cryogenic temperatures in several fields of applied research because of the relevance to (cryo) preservation and bioprotection of cells, tissues, therapeutic proteins and food.5,6 A significant motion that manifests itself in biological systems (proteins, membranes, lipidprotein complexes) at cryogenic temperatures, and that can drive functionally important molecular processes at physiological temperatures, is the librational motion. It consists of fast harmonic oscillations in the nanosecond timescale, of small angular amplitude around an equilibrium position. Evidence for such a motion has been obtained by linear and non-linear continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy in spin-labelled proteins.7-10 Librational oscillations have been described by Dzuba and coworkers for a variety of spin-probes in glassy media.11,12 Full characterization of the librational dynamics via determination of the mean-square angular amplitude, 〈 〉, and rotational correlation time, , of the motion is obtained by combining spin-label CW-EPR and two-pulse electron spin echo detected (ED) spectra.12,13 This approach is based on the fact that EDspectra depend on the amplitude-correlation time product, 〈 〉 , whereas CW-EPR spectra allow independent measurement of the mean-square amplitude, 〈 〉, of the librations. Thus, by considering the results from both techniques, the rotational correlation time of the motion can be evaluated. In this way, librational oscillations have been detected and characterized in proteins,14 in natural membranes and their extracted lipids,15,16 in monounsaturated phospholipid bilayers,17 and in membranes containing high cholesterol,13 as well as in lipid-peptide complexes.18 Strictly related to low-temperature librational motion is the glass-like behaviour of biosystems, in that angular librational fluctuations of appreciable amplitude set in around 200 K, where the dynamical or glass transition takes place in macromolecules and supramolecular aggregates. Scattering and dielectric relaxation studies are used to characterize the glassy behavior of nucleic acids,19 proteins, 20-26

and membranes and lipid bilayers.2, 27 Additionally, in membrane model systems of various lipid 3 ACS Paragon Plus Environment


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compositions, the glass-like behavior is investigated by using differential scanning calorimetry,28,29 Raman spectroscopy,4,30 and spin-label EPR. 4,31 In the present work, we use CW-EPR and ED-spectra of phosphatidylcholine spin-labelled at position C5 and C16 along the sn-2 chain (5- and 16-PCSL) to characterize the librational dynamics of cryogenically frozen membranes that are formed by the ester-linked lipid dihexadecanoyl phosphatidylcholine (DPPC) and by the analogue lipid with ether-linked chains dihexadecyl phosphatidylcholine (DHPC) (see Figure 1).

Figure 1. Chemical structures of the lipids DPPC and DHPC and the spin-labelled lipids 5- and 16PCSL used in this study.

Ester-lipids are the most abundant in eukaryotic and bacterial membranes and are widely used to form membrane model systems.32 Ether-lipids occur in minor amounts in eukaryotes and bacteria but are omnipresent in the membranes of the Archaea, where they promote membrane stability and contribute to the ability of the organism to survive extreme habitats.32,33 Both DPPC and DHPC lipids at full hydration form lamellar membranes that differ in the (gel) low-temperature phases: while noninterdigitated bilayers are formed by DPPC, the chains are interdigitated in DHPC dispersions.34-36 Here we present and discuss how the different lipid packing influences the characteristics of librational motion and the dynamical transition of DPPC and DHPC model membranes in the frozen state over the temperature range 77-260 K.

EXPERIMENTAL SECTION

Materials. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and deuterium oxide (99.9 atom% 2

H) were obtained from Sigma/Aldrich (St. Louis, MO). 1,2-di-O-hexadecyl-sn-glycero-34 ACS Paragon Plus Environment

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phosphocholine (DHPC) and the spin-labeled lipids 1-palmitoyl-2-(n-(4,4-dimethyl-oxazolidine-Noxyl)stearoyl)-sn-glycero-3-phosphocholine (n-PCSL with n = 5, 16) were obtained from Avanti Polar Lipids (Alabaster, AL). All materials were used as received without further purification.

Sample Preparation. DPPC or DHPC and 0.5 mol% n-PCSL spin label were codissolved in methanol/chloroform (1:2 v/v). The solvent was first evaporated under a nitrogen gas stream and then under vacuum overnight. The dried thin lipid films were then fully hydrated (final lipid concentration 50 mM) in D2O by heating at 50 °C and periodically vortexing. The hydrated samples were finally concentrated by centrifugation at 3000 rpm for 30 min by using a benchtop centrifuge, the excess aqueous supernatant removed and the pellets were loaded in quartz tubes (ID 3 mm) for EPR measurements.

CW-EPR Spectroscopy. Conventional CW-EPR spectra were acquired on a Bruker ESP-300 spectrometer operating at 9 GHz with 100-kHz field modulation and equipped with an ER4201 TE102 standard rectangular cavity and with an ER 411VT temperature controller (both from Bruker). For measurements at 77 K, the spin-labelled lipid samples were rapidly frozen in liquid nitrogen and then measured in a finger dewar containing liquid nitrogen.

Pulsed EPR Spectroscopy. Echo-detected EPR spectra were acquired on an ELEXSYS E580 9-GHz Fourier Transform FT-EPR spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with an MD5 dielectric resonator and a CF 935P cryostat (Oxford Instruments, UK). The spin-labeled lipid samples were rapidly frozen in liquid nitrogen, transferred to the resonant cavity that was pre-equilibrated at 77 K and measured on heating. Primary, two-pulse ⁄2 − − − − ℎ echo-detected (ED)-EPR spectra were obtained by recording the integrated spin−echo signal at fixed interpulse delay , while sweeping the magnetic field. The microwave pulse widths were 32 and 64 ns, with the microwave power adjusted to provide ⁄2 and -pulses, respectively. The magnetic field was set to the EPR absorption maximum, and the integration window was 160 ns. The original ED-spectra, 2, , were corrected for instantaneous spin diffusion arising from static spin-spin interaction by using spectra recorded at 77 K, where motional contributions are



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negligible. The corrected spectra, 2, , recorded at temperature T are plotted as a function of magnetic field, H, according to:37 2, = 2,

, ,

(1)

where ! is the shortest value of for which ED-spectra were obtained. Relaxation rates, ", # , , were determined from the ratio of corrected ED-spectra recorded at two different values, # and , of the interpulse delay by using the following relation:13 ,

", # , = ln &' , ) ∙ (

#

( +'

(2)

where 2, is the ED-spectral lineheight at field position H. Data reproducibility was assessed by repeating the measurements.

RESULTS CW-EPR measurements. CW-EPR spectra of 5- and 16-PCSL in DPPC and DHPC membranes at selected temperatures from 150 to 260 K are shown in Figure 2.


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Figure 2. CW-EPR spectra at different temperatures of 5- and 16-PCSL in DPPC noninterdigitated membranes and in DHPC interdigitated membranes. Spectral width = 110 Gauss. The spectra of 5-PCSL in noninterdigitated DPPC frozen bilayers display a large anisotropy, typical of spin-labelled lipids immobilized on the conventional EPR timescale. A limited decrease of the spectral anisotropy is only evident at the highest temperatures. Moreover, the powder patterns are characterized by inhomogeneously broadened lines of Gaussian character which narrow progressively with increasing temperature until they become almost purely Lorentzian (see resonance lines at low-field). The spectra of 16-PCSL in DPPC are also characteristic powder patterns of immobilized lipids. However, at any temperature they display a considerable lower anisotropy and narrower lines than those of 5-PCSL. Highly anisotropic spectra are also recorded for both 5- and 16-PCSL in DHPC membranes with interdigitated chains. Different from what is seen in DPPC bilayers, both positional isomers show almost similar spectral widths when the spectra are compared at the same temperature. Only at the highest temperatures, the spectral anisotropy of 16-PCSL decreases more rapidly than that of 5-PCSL. Quantitatively, the temperature variations of the spectral anisotropy of the spin-labelled lipids 5and 16-PCSL in DPPC and DHPC membranes are obtained from the plots of the motionally averaged 7 ACS Paragon Plus Environment


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hyperfine splitting, 2〈,-- 〉, as a function of temperature as reported in Figure 3. 2〈,-- 〉 corresponds to the separation of the two outer peaks in the EPR spectra (see Figure 2).

Figure 3. Temperature dependence of the motionally averaged hyperfine splitting, 2〈,-- 〉, for 5(squares) and 16-PCSL (circles) in DPPC noninterdigitated membranes and DHPC interdigitated membranes. For DPPC bilayers (Figure 3, upper panel), high 2〈,-- 〉-values of about 68 Gauss are obtained for 5PCSL which decrease slightly and moderately to ca. 66 Gauss only on approaching the highest temperature. In contrast, considerably lower values are obtained for 16-PCSL over the whole temperature range. 2〈,-- 〉 for 16-PCSL in DPPC shows a limited decrease from ca. 64 to ca. 63 Gauss up to 200-220 K, followed by a marked reduction beyond 220 K. The temperature dependence of the motionally averaged hyperfine splitting for DPPC, in which 2〈,-- 〉 (16-PCSL) < 2〈,-- 〉 (5-PCSL), is diagnostic of bilayer membranes with noninterdigitated chains.

It indicates that the two membrane regions probed by the nitroxide labels are characterized by different mobility. The first -CH2- methylene segments in proximity to the polar/apolar interface, which is sampled by 5-PCSL, is a bilayer region of restricted mobility (i.e., high 2〈,-- 〉), whereas the terminal CH3 methyl ends of the chain, which are sampled by 16-PCSL, are characterized by higher mobility (i.e., low 2〈,-- 〉). 38,39 8 ACS Paragon Plus Environment

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Another reason for the behavior in the upper panel of Figure 3 arises from the differences in the polarity: the interfacial hydrocarbon region is a high-polarity zone (i.e., high ,.. -value), in contrast to the deep interior that is characterized by a much lower environmental polarity (i.e., low ,.. -value).40 For DHPC interdigitated membranes (Figure 3, lower panel), 2〈,-- 〉 for both labels has a high value of ca. 69 Gauss, and is temperature-independent below 230 K for 5-PCSL and below 220 K for 16-PCSL. Above these temperatures, 2〈,-- 〉 decreases more rapidly for 16- than for 5-PCSL. The EPR results in DHPC are typical of chain interdigitation. Indeed, when interdigitated gel phase membranes are studied by EPR of chain-labelled lipids, a positional isomer at the chain termini (viz., 16-PCSL) is located near the interfacial region on the opposite side of the lamellae, and is motionally restricted to an extent comparable to that of a positional isomer at the first segments of the lipid chains (viz., 5-PCSL).38,41 The data also indicate that in the interdigitated DHPC phase both ends of the chains are located in the high-polarity interfacial region and are exposed to the solvent.

Pulsed EPR measurements. Two-pulse, echo detected ED-EPR spectra of 5- and 16-PCSL in DPPC and in DHPC membranes recorded at 200 K for different values of the interpulse separation time, , and corrected for instantaneous diffusion according to eq 1, are shown in Figure 4.



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Figure 4. Two-pulse echo-detected ED-EPR spectra at 200 K at different interpulse separation time for 5- (left-hand side) and 16-PCSL (right-hand side) in DPPC noninterdigitated membranes (upper panel) and DHPC interdigitated membranes (lower panel). Solid lines are the corrected experimental spectra and dashed lines are simulations for isotropic librational motion. Beneath each set of ED-spectra is given the anisotropic part of the relaxation rate, W, obtained according to eq 2 from pairs of ED-spectra with interpulse separations of # = 168 and = 296 ns, # = 168 and = 424 ns, or # = 168 and = 552 ns. 10 ACS Paragon Plus Environment

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In any sample, the dependence of the ED-lineshape on echo-delay, , reveals preferential spin relaxation in the intermediate spectral regions at low and high field that is characteristic of librational dynamics.12,13,37 The occurrence of librational motion in DPPC and DHPC lipid membranes is also confirmed by spectral simulation. By comparing the experimental and the simulated spectra in Figure 4 (solid and dashed lines, respectively), it can be seen that the ED-spectra are well described with the model used previously for chain-labelled lipids in membranes that is known as the “isotropic” model for librations, in which uncorrelated librational motions take place simultaneously around the three x-, y-, and z- nitroxide axes.13,37 The anisotropic curves of the relaxation rate, W, obtained as defined by eq 2 are given underneath the corresponding ED-spectra in Figure 4. W-relaxation spectra evaluated for different pairs of # and coincide to within the noise level. This shows that the relaxation is close to exponential and confirms the validity of the isotropic model for libration. The relaxation rates are characterized by the maximum values, WL and WH, determined in the low- and high-field regions, respectively, of the ED-spectra (see Figure 4). The difference in intensity at the two positions arises simply from the different inherent sensitivities of the low- and high-field spectral regions to spin relaxation. The Wvalues collected from the ED-spectra at different temperatures are used to determine the motional parameter 〈 〉 and to characterize the librational motion of DPPC and DHPC membranes, as shown below.

Characterization of the librational motion. The upper panel of Figure 5 shows the temperature dependence of the librational amplitude-correlation time product, 〈 〉 , of 5- and 16-PCSL in DPPC and DHPC membranes. The values are obtained from the measurements of the relaxation parameter at low-field, WL, by using a conversion factor relating 〈 〉 to WL of 1.41x1017 rad-2s-2 for 5-PCSL and of 1.05x1017 rad-2s-2 for 16-PCSL, as established previously.13 The intensity of librational motion for both spin-labels in each lipid membrane increases with the temperature. However, interesting differences between DPPC and DHPC are evident from Figure 5. In DPPC noninterdigitated bilayers, the motional parameter 〈 〉 increases, first slowly for temperatures up to 180 K and then more rapidly for T > 180 K. In DHPC membranes with interdigitated acyl chains, 〈 〉 increases linearly and slowly up to 165-180 K, and then more steeply compared to DPPC for T > 180 K. 11 ACS Paragon Plus Environment


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Figure 5. Temperature dependence of the (upper panel) amplitude-correlation time product, 〈 〉 , (middle panel) mean-square amplitude, 〈 〉, and (bottom panel) correlation time, , of libration for 5(squares) and 16-PCSL (circles) in DPPC noninterdigitated membranes and DHPC interdigitated membranes.

The temperature dependence of the mean-square angular amplitude, 〈 〉, of librational motion for 5- and 16-PCSL in DPPC and DHPC is given in the middle panel of Figure 5. This parameter is derived from the partially averaged hyperfine splitting, 2〈,// 〉, of the CW-EPR spectra according to the equation:12,42 〈 〉 =

011 +〈,22 〉 011 +033

(3)

where the angular brackets indicate a motionally averaged hyperfine tensor (of principal elements ,44 , ,55 and ,.. ). In DPPC bilayer membranes (Figure 5, left middle panel), the librational amplitude increases with temperature. For 5-PCSL the variations are small and become more evident for temperatures higher than 220 K. For 16-PCSL, the mean-square amplitude is higher than that of 5-PCSL and increases with temperature, first slowly and then more rapidly for T > 200 K. For 5-PCSL (16-PCSL) the root-mean12 ACS Paragon Plus Environment

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square angular amplitude changes from 5.5±1.3° (7.7±1.0°) to 5.9±1.6° (9.7±1.1°) and to 10.1±0.7° (18.6±0.6°) when the temperature is varied from 150 K to 200 K and to 260 K. In DHPC interdigitated membranes (Figure 5, right middle panel), the angular amplitude of libration is small up to 240 K for 5-PCSL and up to 220 K for 16-PCSL, and then increases rapidly, more for 16than for 5-PCSL. At 260 K, 〈〉 reaches the values of 9.3±0.7° and 17.0±0.5° for 5- and for 16-PCSL, respectively. In the lower panel of Figure 5 is shown the rotational correlation time, , for the librational motion of the chain labelled lipids in DPPC and DHPC membranes. is determined by dividing the 〈 〉 values deduced from ED-spectra by the independent determinations of 〈 〉 obtained from the CW-EPR spectra. From Figure 5, it can be seen that the -values lie in the subnanosecond timescale for DPPC bilayers and in the nanosecond timescale for DHPC interdigitated membranes and are lower for 16PCSL than for 5-PCSL. The determinations of are affected by large errors also because echo intensities become progressively weaker. Note that the echo disappears at a lower temperature in DHPC than in DPPC (see Figure 5).

DISCUSSION Fast torsional librational oscillations of small amplitude have been detected in the temperature range 77-260 K in bilayer membranes of the diacyl, ester-linked DPPC lipid with noninterdigitated chains, and in membranes of the dialkyl, ether-linked DHPC lipid with interdigitated chains. An interesting feature that emerges from the CW- and ED-EPR results of our study is that the different molecular chain packing of DPPC and DHPC membranes (noninterdigitated vs. interdigitated chains) affects the characteristics of the librational motion. In DPPC frozen bilayer membranes the angular librations are in the subnanosecond timescale and their amplitude increases with temperature. Moreover, the angular amplitude and the rotational correlation time of the librational motion depend on the label position, n, along the DPPC chain: the amplitude becomes larger and the motion becomes more rapid on moving from the polar/apolar interface towards the bilayer midplane. These results are expected for lipid bilayer assemblies and are in agreement with previous EPR results on bilayers made of lipids both synthetic and extracted from natural membranes.13,16,17,43 The behavior of 〈 〉 in DPPC bilayers in Figure 5 mirrors that of 2〈,-- 〉 in 13 ACS Paragon Plus Environment


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Figure 3 in which the decrease of the outer hyperfine splitting with temperature and with n corresponds to the increase of the amplitude of torsional librations. The packing with interdigitated chains in DHPC lamellae leads to librational motion with rotational correlation time on the nanosecond timescale. At low temperature, the 〈 〉-values are small and temperature and label-position independent. Only on entering the higher temperature regime, the angular amplitudes increase and are larger at the chain termini than at the beginning of the chain. The behavior of the chain-labelled lipids in DHPC membranes is consistent with the interdigitated phase in which the positional isomers at the chain termini are motionally restricted to an extent comparable to those in proximity of the polar/apolar interface.38,41 At high temperature, it is likely that 16-PCSL acquires significant freedom of motion relative to 5-PCSL, since it is located in the interfacial region where the polar heads are spaced apart by interdigitation. In the temperature dependence of the mean-square amplitude of the spin-labelled lipids in DPPC and DHPC lamellar phases in Figure 5, a considerable increase of the librational amplitude is clearly evident for T > 200 K for DPPC and T > 220 K for DHPC. This trend was also observed previously by EPR for the librational motion of a variety of systems: spin labels of different geometrical shape in glass-forming solvents,12,44 chain-labelled lipids in membranes, 13,16,17,31 and spinlabelled proteins.10,14-16,45-47 Most significantly, a behavior similar to the temperature dependence of 〈 〉 of the librational motion that is recorded by EPR is found for the mean-square atomic displacements, , measured in molecular glasses and various biosystems by dielectric relaxation and scattering techniques.2,20,21,23,24, 26,27

In all cases, a rapid increase in amplitude of the atomic motions is found at the dynamical or glass

transition temperature, Td, of the systems around 200 K. From the data of the present study, a dynamical transition associated with increased torsional lipid chain fluctuations can be detected at Td ≈ 220 K in DPPC and at ≈ 240 K in DHPC labelled with 5PCSL and at slightly lower temperature at ≈ 210 K and 230 K, respectively, with 16-PCSL. The dynamical transition can be inferred from the rapid increase in 〈 〉, or from the decrease in 2〈,-- 〉 (see Figure 3), and also from the conversion from inhomogeneous Gaussian line broadening to homogenous Lorentzian line broadening of the CW-EPR spectra with temperature (see Figure 2).48 Moreover, in DPPC bilayers a moderate increase in intensity of motion and slight increase in 〈 〉 at ca. 150 K is evident that can be ascribed to activation of methyl group rotations in the label.49 Such a transition is not observed in the interdigitated DHPC membranes. 14 ACS Paragon Plus Environment

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Our results are in agreement with a number of studies on lipid membranes. A dynamical transition was reported by using neutron scattering in hydrated purple membrane at Td ≈ 200 K

2

and in

multibilayers of dimyristoylphosphatidylcholine at Td ≈ 230 K;27 in the latter, the transition of the polar heads sets in at a higher temperature than that of the hydrocarbon tails. Further, a dynamical transition was revealed by Raman scattering in lipid bilayers with saturated and unsaturated chains and occurred near 200 K in DPPC bilayers.4,30 A glass-like behavior was observed by differential scanning calorimetry in membranes of tethaether lipids with methyl-branched chains from Thermoplasma acidophilum with the transition temperature over the 180-225 K range,28 and in long-chain phosphatidylcholine mesophases.29 When above the transition, i.e., for T > Td, the temperature dependences of 〈 〉 for 5- and 16-PCSL in DPPC and DHPC conform with an Arrhenius law: 〈 〉 ≈ 〈 〉! 78−9 ⁄:; and are reasonably well described with a single activation energy, 9 . The values of 9 are 15.0±1.5 kJ/mol and 13.0±0.3 kJ/mol for 5- and 16-PCSL in DPPC noninterdigitated bilayer membranes, whereas higher values of 50±7 kJ/mol and 32±1 kJ/mol are found for 5- and 16-PCSL in DHPC interdigitated lamellar phase. These results indicate that the interdigitated packing of the hydrocarbon chains confers to DHPC membranes higher stability and a higher activation energy is required for the onset of stochastic librations of the lipid molecules. Moreover, the motion of the water molecules in close proximity of the membrane surface may contribute to the activation energy. Such an effect may be more relevant in DHPC interdigitated membranes. The 9 -values in DPPC are comparable to previous determinations based on the dependence on temperature of the phase-memory time of the electron spin-echo decays for n-PCSL in bilayers of DPPC+50 mol% cholesterol.43 In addition, by applying similar analysis to other biomembranes studied previously, values of 9 comparable to those in DHPC have been found for chain-labelled lipids in membranes of Na,K-ATPase16 and for TOAC-8 spin-labelled alamethicin introduced into phosphatidylcholine bilayers.18 The 9 values for spin-labelled membranes determined in the present study are in the range estimated (9 ≈ 20-40 kJ/mol) for a number of proteins under different solvent conditions by using various physical methods3,21,47 exploring the glass behaviour of the biosystems on different timescale.

CONCLUSIONS The results of this study establish and characterize the segmental lipid chain librations in lowtemperature membrane phases, both in bilayers of DPPC and in the interdigitated DHPC lamellae. 15 ACS Paragon Plus Environment


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They provide insight into the glassy behavior of the lipid assemblies. Thus spin-label EPR proves to be a useful tool for investigating not only the low-temperature dynamics but also the glass-like properties of a wide variety of biosystems. In bilayer membranes of the ester-linked diacyl DPPC lipid, the librations are on the subnanosecond timescale and the angular amplitude increases with temperature and are larger at the chain termini than at the beginning of the chain. Compared to DPPC, in membranes of the ether-linked dialkyl DHPC lipid with interdigitated chains, the librations are on a slower timescale and restricted to small angular amplitude at both chain positions of labeling at low temperature. Only at high temperature appreciable amplitude comparable to that in DPPC is attained. In the frozen state, the membrane environment with ester- and ether-linked apolar chains to the glycerol backbone has similarities to the glassy state found in other biosystems. The dynamical transition in the lipid lamellae is in the range 210-240 K and occurs at higher temperature in the interdigitated DHPC membranes than in DPPC. The activation energy for the onset of stochastic large-amplitude motions is higher in DHPC than in DPPC. Our results on the low-temperature properties of the lipid membranes (i.e., librational motion and glasslike behavior) may be important in cryoprotection and in the structure-dynamics-function relation of biosystems. Glass formation by aqueous solutions has been implicated in biological cryopreservation5 and the glassy behavior of lipid membranes may affect the cryopreservation of proteins and supramolecular complexes. The librational motion is clearly investigated at low-temperature, when large-scale rotations are frozen out. It inevitably occurs also at higher, physiological temperature, where the slower, large-scale rotations make difficult to resolve the fast librations explicitly. The rapid, small-amplitude angular oscillations detected in lipid lamellae in the present study may affect the functional behavior of cell membranes and the activity of membrane-embedded integral proteins.


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Low-Temperature Dynamics of Chain-Labeled Lipids in Ester- and

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