Dynamical Transitions at Low Temperatures in the Nearest Hydration

Jan 12, 2017 - Physics Department, Novosibirsk State University, Novosibirsk ... ESEEM frequency spectra resemble the solid-state 2H NMR line shape th...
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Dynamical Transitions at Low Temperatures in the Nearest Hydration Shell of Phospholipid Bilayers V. N. Syryamina†,‡ and S. A. Dzuba*,†,‡ †

Institute of Chemical Kinetics and Combustion, Russian Academy of Sciences, Novosibirsk 630090, Russian Federation Physics Department, Novosibirsk State University, Novosibirsk 630090, Russian Federation

‡

ABSTRACT: For the so-called dynamical transition from harmonic to anharmonic (or diffusive) motions in biological systems, the presence of hydration water is important. To explain the molecular mechanism of this transition, the information on molecular motions in the nearest hydration shell would be helpful. In this work, to study molecular motions in the nearest hydration shell of spin-labeled model biological membranes, a pulsed version of electron paramagnetic resonance, electron spin echo envelope modulation (ESEEM) spectroscopy, is used. For hydration by deuterium water, the 2H ESEEM frequency spectra resemble the solid-state 2H NMR line shape that is widely used for structural and dynamical studies. Two types of model membranes were investigated and compared: bilayers consisting of unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and bilayers consisting of fully saturated lipid 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC). The lipid chain packing for the POPC bilayer is known to be more defective than that for the DPPC bilayer. For both the POPC and the DPPC bilayers, the 2H ESEEM NMRlike spectra showed a sharp narrowing between 180 and 190 K. From the other side, in both bilayers at 188 K, an inflection was observed for the temperature dependence of molecular motions detected by the spin relaxation of spin labels in the bilayer interior. It was concluded that dynamical transition in the bilayer interior is accompanied by an onset of isotropic water molecular dynamics in the nearest hydration shell of the bilayer with a rate of ∼105 s−1. Also, the 2H ESEEM NMR-like spectra in the POPC bilayer showed slight changes above 100 K that could be ascribed to another dynamical transition resulting in the appearance of restricted orientational motion of water molecules. These data also are interrelated with spin relaxation of spin labels in the POPC bilayer interior and support the hypothesis ascribing the transition at 100 K to excessive lipid chain flexibility.

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INTRODUCTION

theory or model of the dynamics, even on a qualitative phenomenological level.1−5 This dynamical transition correlates with a transition in the hydration shell of the biomolecules.3−5,21−24 Water may act as a plasticizer in the motions of the hydrated proteins3 and the dynamics of the biological macromolecules. The alternative idea is that the dynamics of biological macromolecules and their hydration water are both determined by the chemical and three-dimensional structures of the biomolecules.24 Regardless, it is clear that the mutual influence of biomolecules and their hydration water must be considered. These and other aspects of water−protein dynamic coupling have intensively been discussed in the literature, see, for example, reviews.3−5 To find the relationship between the dynamical transition in the biomolecules and in the surrounding hydration shell, data on motions should be separately obtained in both media. To attain this goal, either the biomolecules or the water molecules may be deuterated in neutron-scattering experiments10,11,25,26 (only hydrogens are visible in these experiments). The data

Water plays an important role in the structure, dynamics, and function of biomolecules. In biological media, the so-called dynamical transition, which is observed by the Mössbauer absorption, neutron scattering, and molecular dynamics (MD) simulations,1−5 occurs only for hydrated biomolecules. This transition manifests itself as an inflection of the atomic meansquare displacement (MSD) temperature dependence at 170− 230 K, which is due to the transition from a harmonic to an anharmonic (or diffusive) motion. It is observed for different kinds of biological systems: proteins,1−5 DNA and RNA,6 small amino acids,7 biological membranes,8−13 as well as nonbiological polymers and nonaqueous solvents.14 Other experimental techniques such as dielectric relaxation,15 infrared and NMR spectroscopy,16 Raman spectroscopy,17,18 and spinlabeled pulsed electron paramagnetic resonance (EPR)19,20 also demonstrated this dynamical transition. However, despite intensive investigations of this phenomenon, a general atomistic picture of internal biomolecular dynamics is still missing, and a classification of different relaxation processes and their connection to biological function and activity are still needed.4,5 In addition, there is no accepted © XXXX American Chemical Society

Received: October 6, 2016 Revised: January 11, 2017 Published: January 12, 2017 A

DOI: 10.1021/acs.jpcb.6b10133 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

glycero-3-phosphocholine (POPC) and bilayers consisting of fully saturated lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). It is known that the lipid chain packing for the POPC bilayer is more defective than that for the DPPC bilayer.18,20 In both cases, bilayers contained spin-labeled lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(TEMPO)choline (Tempo-PC) possessing a spin label Tempo at the lipid polar head. Tempo-PC is assumed to be incorporated into the bilayer like other lipids, with the Tempo residue located on the bilayer surface. For comparative purposes, molecular glass prepared by the mixture of deuterated water and dimethylsulfoxide (DMSO) and containing nitroxide spin-probe Tempone was also investigated.

analysis obtained in this way shows that hydration water suppresses protein motions at lower temperatures (≤200 K) and facilitates protein dynamics at higher temperatures. However, neutron scattering does not provide a microscopic mechanism of the hydrogen atomic motion. In addition, the solid-state 2H NMR may be applied for deuterated biological media.27−32 The advantage of NMR is that it is capable of providing information on the mechanism of the molecular motion of water. However, 2H NMR probes molecular motions in the whole water volume, including both the nearest hydration shell and the more remote water molecules. Therefore, the 2H NMR data generally demonstrate the remarkable heterogeneity in motions of the hydration water.27−32 The application of a low hydration level does not solve the problem because it introduces a hydration environment that is not characteristic of the protein environment in the living systems.4 It is noteworthy that for directly probing the structure and dynamics of the nearest hydration shell, different experimental techniques have been developed, such as dynamic nuclear polarization (DNP),33−36 NMR relaxation dispersion,37 and infrared spectroscopy.38 However, as these techniques are applied at an ambient temperature, they cannot be used for studying the dynamical transition phenomenon when the measurements are carried out across a wide temperature range. The recently suggested approach39 based on electron spin echo envelope modulation (ESEEM)40 spectroscopy, the version of pulsed EPR, is capable of studying orientational dynamics in the hydration shell of a spin label in the biomolecules. The ESEEM effect is produced by the electron−nuclear hyperfine interaction (HFI) between the unpaired electron of the spin label and the surrounding matrix nuclei; for deuterium nuclei, it is determined by a combination of HFI and nuclear quadrupole interaction (NQI).40 When the NQI contribution dominates, the 2H ESEEM spectra resemble the 2H NMR line shape.39 As compared with neutron scattering and solid-state NMR, the advantage of the suggested ESEEM approach39 is the possibility to study motions in the nearest hydration shell, at distances between 0.5 and 1 nm from the spin label in the biomolecule41 and for a fully hydrated sample. Also, ESEEM provides information on the mechanism of water molecular motion, like in solid-state NMR. As compared with DNP, the advantage is the possibility to study orientational dynamics at low temperatures in the vicinity of the dynamical transition. In addition to the inflection of MSD temperature dependence seen at 170−230 K in biological systems, a similar inflection was also observed at 100−150 K, which is less pronounced but well detected. This transition is commonly attributed to the thermally activated motion of methyl groups.11,42−44 An alternative hypothesis drawn from the analysis of Raman scattering in the phospholipid bilayers18,20 is that this transition could occur because of an onset of excessive lipid chain flexibility at 100 K. To explain the nature of transition near and above 100 K, alternative experimental approaches would be helpful as well. Here, 2H ESEEM is used for studying the motions of deuterated water molecules near the spin-labeled phospholipid bilayer surface in a temperature range between 80 and 220 K. The use of deuterated water and normal protonated lipids allows to refine motional effects belonging to water exclusively. Two types of model membranes were investigated: bilayers consisting of monounsaturated lipid 1-palmitoyl-2-oleoyl-sn-

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EXPERIMENTAL SECTION Substances and Sample Preparation. The spin-labeled lipid Tempo-PC, monounsaturated lipid POPC, and saturated lipid DPPC were purchased from Avanti Lipids and used as received. The stable nitroxide 2,2,6,6,-tetramethyl-4-oxo-piperidinyl-1-oxyl (Tempone) was purchased from Sigma and purified by sublimation. Deuterated water and (nondeuterated) DMSO were purchased from Sigma Aldrich and used without further purification. Lipid bilayers were prepared as multilamellar vesicles using the following process. The lipid POPC or DPPC with spinlabeled lipid Tempo-PC at a molar ratio of 100:1 was dissolved in chloroform and evaporated under the nitrogen stream. The obtained thin lipid film was placed under vacuum for 4 h. Then, it was hydrated by deuterated water for 4 h at 5 °C for POPC/ Tempo-PC and at 50 °C for DPPC/Tempo-PC and thereafter placed into an ultrasound bath for 20 min. The hydration level, h, was ∼1 w/w (water/lipids). After hydration, the POPC/ Tempo-PC sample was quickly frozen in liquid nitrogen, and the DPPC/Tempo-PC sample was stored at 5 °C for 12 h, then at −5 °C per night, and finally it was frozen in liquid nitrogen. Tempone was dissolved in the water/DMSO mixture (60/40 w/w) at a concentration of about 10−3 M. The samples of the water/DMSO mixture with Tempone upon freezing appeared in the form of transparent glasses. EPR and ESEEM Measurements. Continuous wave (CW) EPR experiments were carried out by an X-band Bruker E380 EPR spectrometer using a dielectric Bruker ER 4118 X-MD-5 cavity and an Oxford Instruments CF-935 cryostat. EPR spectra were recorded at a modulation amplitude of 0.05 mT and a modulation frequency of 100 kHz. The microwave power was set low enough to avoid spectra saturation. ESEEM experiments were carried out by an X-band Bruker ELEXSYS E580 EPR spectrometer using a split-ring Bruker ER 4118 X-MS-3 cavity and an Oxford Instruments CF-935 cryostat. The threepulse microwave sequence used was π/2−τ−π/2−T−π/2−τ− echo, and the four-pulse sequence was π/2−τ−π/2−T/2−π− T/2−π/2−τ−echo. The microwave pulse width was 16 and 32 ns for the π/2 pulse and the π pulse, respectively. The time delay, τ, was set to 200 ns; this is close to the third blind spot for the 1H matrix ESEEM, but it provides the maximum intensity for the 2H matrix ESEEM.40 Unwanted echoes were removed by a four-step phase cycling program.40 For each experiment, the time delay, T, was increased from Tmin = 248(496) ns for the three(four)-pulse experiment up to Tmax, where Tmax was either 8 μs (for the POPC and DPPC bilayers) or 12 μs (for the water/DMSO mixture) with a time step of 16 ns. The magnetic field was set to a maximum of the echodetected EPR spectrum. B

DOI: 10.1021/acs.jpcb.6b10133 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B The cavity in both the CW EPR and ESEEM experiments was cooled by gaseous nitrogen with an accuracy of ±0.5 K. A thermocouple placed into the cavity was used to monitor the sample temperature. ESEEM Data Treatment. To separate echo decay V(2τ,T) due to the spin relaxation and ESEEM effect, the standard procedure was performed41,45 with the smoothing decay function ⟨V(2τ,T)⟩ considered as a third-order polynomial in a semilogarithmic scale. The normalized ESEEM curve, Vn(t), where t = τ + T, was obtained by using the following expression Vn(2τ , T ) =

V (2τ , T ) −1 ⟨V (2τ , T )⟩

(1)

A computer program was written (the MATLAB software) to perform the modulus Fourier transformation of the obtained Vn(2τ,T) dependence, taken as a function of τ + T. The Fourier transform for all samples was obtained using a Gauss-window function.

Figure 2. Time-domain (left) and frequency-domain (right) 2H ESEEM spectra for the glassy water/DMSO mixture. Data are shifted along the vertical axis by a dimensionless value of 1.0 (left) and by 2 MHz−1 (right). Spectra are normalized to the unity value of their total intensity in the frequency range of 1.6−2.8 MHz. The dotted lines show the spectral positions of singularities at 72 K.

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RESULTS CW EPR spectra were typical for immobilized nitroxide spin labels. Figure 1 shows temperature dependence of the spectral

Figure 1. Temperature dependence of the spectral splitting 2AZZ in the EPR spectra. The inset shows how the spectra change with temperature and how the splitting is measured (the sample is the POPC bilayer).

Figure 3. 2H ESEEM spectra for the POPC (left) and DPPC (right) bilayers. Spectra are normalized to the unity value of their total intensity in the frequency range of 1.6−2.8 MHz and consequently shifted along the vertical axis by 2 MHz−1. The dotted lines show the spectral positions of singularities at 78 K for the POPC bilayers and at 100 K for the DPPC bilayers (see text for details).

splitting 2AZZ obtained for all the samples. One can see that 2AZZ is nearly temperature independent below 250 K for the bilayers and below 200 K for the water/DMSO mixture. The decrease of 2AZZ above these temperatures implies onset of the overall orientational motion of spin labels that becomes visible in the CW EPR time scale (correlation time of motion is shorter than 10−7 s). The electron spin echo signal could be well detected at temperatures up to 200−220 K for the bilayers and up to ∼160 K for the water/DMSO mixture (the signal disappears at higher temperatures because of the fast spin relaxation). Figure 2 (left panel) presents the time-domain 2H ESEEM data for the glassy water/DMSO mixture. The data were pretreated using eq 1. Figure 2 (right panel) presents the Fourier transforms, that is, the ESEEM frequency spectra. For the POPC and DPPC bilayers, the ESEEM frequency spectra are shown in Figure 3. The spectra in Figure 2 and the low-temperature spectra in Figure 3 show the doublets centered at 2.2 MHz, which is the

deuterium Larmor frequency at the X-band EPR spectrometer magnetic field. The doublets may be unambiguously assigned as caused by forbidden EPR transitions that are induced by electron−nuclear interactions with the matrix deuterium nuclei located not closer than 0.5 nm and not farther than 1 nm from the spin label.41,45 The doublet splitting is determined by the sum of HFI and NQI in the absence of motion.39 Some admixture of the broad background line seen in the spectra in Figures 2 and 3 belongs to water molecules directly bonded to the spin label.41,45 Previously, it was found39 that the four-pulse ESEEM experiment allows to eliminate the HFI contribution to the 2 H ESEEM frequency spectra, refining the pure NQI contribution. When HFI is of the same order as NQI (it takes place for nitroxides with the deuterated methyl groups39), C

DOI: 10.1021/acs.jpcb.6b10133 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B the three-pulse ESEEM spectra are broader than the four-pulse spectra because of the influence of HFI. In such a case, the fourpulse spectra possess the typical Pake resonance pattern, but this pattern is smeared for the three-pulse spectra.39 However, for the samples studied in this article, the four-pulse ESEEM experiment produced line shapes (data not shown) with the line width nearly the same as for the three-pulse experiment, and the three-pulse spectra possess the nonsmeared typical Pake resonance pattern (see Figures 2 and 3). These observations prove that HFI is small in the case of interaction of the spin label with its surrounding deuterated water, and it does not therefore contribute noticeably to the observed spectral splitting in the ESEEM frequency spectra. So, in this study, we present results of the three-pulse experiment only, assuming in its analysis that the influence of HFI on the spectral shape may be neglected. Spectra in Figure 2 show that the doublet splitting is 0.129 ± 0.005 MHz at 72 K for the water/DMSO mixture. Data in Figure 3 show that this splitting is 0.139 ± 0.005 MHz for the POPC bilayer (78 K) and 0.141 ± 0.005 MHz for the DPPC bilayer (100 K). These values are in good agreement with literature data27−31 on the 2H NMR spectra in frozen interface water in the biological systems, indicating that the doublet splitting is typically between 0.120 and 0.140 MHz. This agreement confirms that the contribution of HFI in the threepulse ESEEM spectral splitting is small and that spectra are mostly determined by NQI. Therefore, the 2H ESEEM spectra observed in this study may be called the 2H ESEEM NMR-like spectra of the surrounded deuterated water. For the water/DMSO mixture, the 2H ESEEM NMR-like spectra shown in Figure 2 (right panel) change only slightly in the whole temperature range studied, even at the temperatures that approach the glass transition temperature, Tg (Tg = 141 K for this composition46). For both the POPC and DPPC bilayers, data in Figure 3 show that the doublet collapses into a single line between 180 and 190 K. This collapse is typical for the 2H NMR spectra47,48 for molecules undergoing an isotropic motion with the rate higher than 105 s−1 (for the POPC bilayer, the spectrum at 180 K might correspond to the intermediate situation when the rate is just of the order of 105 s−1). In Figure 4, the doublet splitting in the 2H ESEEM NMRlike spectra is plotted as a function of temperature for all studied samples. For the POPC and DPPC bilayers, the doublet splitting rapidly disappears above 180 K. For the water/DMSO mixture, the doublet splitting starts to decrease above 137 K, which is close to Tg.

Figure 4. Doublet splitting as a function of temperature for the 2H ESEEM NMR-like spectra, as shown in Figures 2 and 3, for the POPC and DPPC bilayers and water/DMSO mixture. The experimental uncertainty is also shown. The lines are drawn to guide the eye.

deuterated water in the POPC bilayers27 obtained at different temperatures showed that motions become completely frozen at 208 K. However, these data were obtained for a very low water content. For the D2O-hydrated dimyristoyl phosphatidylcholine (DMPC) bilayer, the 2H NMR data29 show about five to six waters near the lipid polar head remain unfrozen at temperatures as low as 200 K. Such a dependence on the water content indicates motional heterogeneity, wherein more mobile molecules are located in the nearest hydration shell. Similar results were also observed for the hydrated proteins.30,31 The 2 H NMR study of water dynamics in the hydrated myoglobin31 has shown noticeable dynamical heterogeneities even at T = 186 K. The hydration water dynamics was studied by 2H NMR32 in bovine serum albumin with hydration level h = 0.26 and in the temperature range 95−233 K. The obtained 2H NMR spectra were separated into the fast and slow spin-lattice relaxation components. The fast component was assumed to correspond to water molecules interacting with protein. The spectra were satisfactorily simulated in the model in which the water molecules underwent a 180° flip. The remarkable narrowing of the 2H NMR line was observed above 200 K, indicating the onset of the isotropic motion of the D2O molecules. The 180° flips of water molecules were found at very lower temperatures (down to 110 K). These results32 are in general agreement with the results of the present work. As seen in Figures 3 and 4, the temperature-induced spectral transformations for the POPC bilayer are more noticeable than those for the DPPC bilayer. Therefore, water motions on the surface of the POPC bilayer are more pronounced. It is not surprising because POPC lipids are more flexible at this temperature.18 Note that data in Figures 2 and 4 show noticeable spectral changes for the reference system of the water/DMSO mixture only above 137 K, which is close to the glass transition temperature, Tg = 141 K. Previously, the analogous difference for the POPC and DPPC bilayers was found for molecular motions inside the bilayers20 when motions of spin-labeled stearic acids incorporated into the bilayer were studied. In the bilayer, the dynamics of stearic acids may be assumed to mimic that of host lipids. As these motions at low temperatures are restricted in angular

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DISCUSSION The orientational motion of water molecules changes the direction of the principal axes of the quadrupole interaction, which has a strong impact on the 2H NMR line shape.47,48 The data in Figure 3 show that the doublet collapses into a single line between 180 and 190 K, and this implies the onset of the isotropic reorientational motion47,48 with correlation time τR < 10−5 s−1. The slight but noticeable spectral transformations seen in Figures 3 and 4 between 100 and 165 K for the POPC bilayer (the hole in the center becomes shallower, and the doublet splitting decreases with an increase in the temperature) are typical for motions restricted in the orientational space.49−51 The obtained 2H ESEEM NMR-like data may be compared with the literature 2H NMR low-temperature data on water hydrating membranes and proteins. The 2H NMR spectra of D

DOI: 10.1021/acs.jpcb.6b10133 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

As clearly seen from data in Figure 5, the transition temperature, Td = 188 K, is close to the temperatures of onset (between 180 and 190 K) for the isotropic water molecular motion, as seen in Figures 3 and 4 for the 2H ESEEM NMR-like spectra. Therefore, it can be concluded that motions in the nearest hydration layer of the membrane and in the membrane lipid core are interrelated. This conclusion is in agreement with numerous studies conducted by using neutron scattering and MD simulation.3−5,21−23,26 The results26 show that above the dynamical transition, the hydration water exhibits diffusive dynamics, whereas the protein motions are localized to