Low-Temperature Molecular Motions in Lipid Bilayers in the Presence

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Low-Temperature Molecular Motions in Lipid Bilayers in Presence of Sugars: Insights into Cryoprotective Mechanisms Konstantin B. Konov, Nikolay P. Isaev, and Sergei Andreevich Dzuba J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp508312n • Publication Date (Web): 08 Oct 2014 Downloaded from http://pubs.acs.org on October 15, 2014

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The Journal of Physical Chemistry

Low-Temperature Molecular Motions in Lipid Bilayers in Presence of Sugars: Insights into Cryoprotective Mechanisms

Konstantin B. Konov,1 Nikolay P. Isaev2 and Sergei A. Dzuba2,3*

1

Zavoisky Physical-Technical Institute, Russian Academy of Sciences, Kazan

420029, Russia 2

Voevodsky Institute of Chemical Kinetics and Combustion, Russian Academy of

Sciences, Novosibirsk 630090, Russia 3

Novosibirsk State University, Novosibirsk, 630090, Russia

*e-mail: [email protected]

1 ACS Paragon Plus Environment


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Abstract Sugars and sugar alcohols can stabilize biological systems under extreme conditions of desiccation and freezing. Phospholipid bilayers solvated by aqueous solutions of sucrose, trehalose and sorbitol at concentrations of 0.2 M and 1 M and containing incorporated spinlabeled stearic acids were studied by electron spin echo (ESE) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR). The phospholipids were 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC), and the stearic acids were labeled by with nitroxide 4,4-dimethyloxazolidine-1-oxyl (DOXYL) attached rigidly at either the 5th or 16th carbon positions. The ratio of the echo time traces for the two field positions in the EPR spectrum possessing the largest and smallest anisotropies gave the anisotropic contribution to the echo decay, which obeys exponential time dependence with good accuracy. At low temperatures, the anisotropic contribution is induced by stochastic (or diffusive) orientational vibrations of the molecule as a whole (i.e., stochastic molecular librations), with the exponential decay rate Wanis proportional to c, where is the mean angular amplitude of the motion and c is the correlation time. In all cases, it was found that Wanis begins to increase sharply above 170 – 200 K, which was ascribed to the dynamical transition known for biological systems at these temperatures. For hydration by the sucrose and trehalose solutions, Wanis was found to increase noticeably also above ~ 120 K, which was explained by bilayer expanding due to direct bonding of sugar molecules to the bilayer surface. The Wanis temperature dependencies were found to be close to those obtained for the simple systems of the nitroxide spin probe TEMPONE in aqueous sorbitol and sugar 1 M solutions. This correlation suggests a possible mechanism of cryoprotective action of sorbitol and sugars due to the similarity of low-temperature motions in the membrane and in the cryoprotectant-containing surrounding liquid.

Keywords: biological membranes, dynamical transition, electron spin echo, spin labels


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Introduction The structures of biological membranes under the extreme conditions of freezing or desiccation can be stabilized by the presence of small sugars and sugar alcohols.1-4 These substances are known to be accumulated by many freeze-tolerant species.3,4 Different molecular mechanisms of this stabilization effect have been proposed. The water replacement hypothesis suggests that sugars replace the hydration water around biomolecules, maintaining their polar groups at the positions close to the native ones even at low temperatures.5–11 Another hypothesis suggests that sugars are mostly excluded from the hydration shell of the membrane and influence only the bulk physicochemical properties of the intercellular liquid.12–18 It was also suggested that, depending on their concentration, sugars may be either bound to or expelled from the hydration shell.18,19 To elucidate the molecular mechanisms of action of cryoprotective agents on biological systems, an examination of molecular motions in hydration shells of biomolecules can also be employed. For instance, based on

31

P and 2H NMR data20,21 it was shown that the presence of

sugars suppresses molecular motions of lipid molecules and increases the temperature at which rotational motion of the lipid headgroup occurs. The results of fluorescence correlation spectroscopy showed a decrease in the lateral mobility of phospholipids hydrated in the presence of sugars.22 Recently, it has been shown23 that the electron spin echo (ESE) technique, which is a pulsed version of electron paramagnetic resonance (EPR) spectroscopy, can also provide useful information on molecular motions in spin-labeled phospholipid bilayers hydrated in the presence of sugars, as it allows investigating molecular motions at low temperatures. ESE detects stochastic (or diffusive) orientational vibrations of the molecule as a whole, – i.e., stochastic molecular librations, with correlation times on the nanosecond timescale.23-28 For these motions, the motional parameter, c, where is the meansquared angular amplitude of motion and c is the correlation time, can be extracted from the



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ESE data. To ensure that echo decays indeed originate from nanosecond stochastic molecular librations, a three-pulse stimulated spin echo23,29,30 technique was employed. Nanosecond stochastic librations most likely31,32 have the same nature as anharmonic motions detected in biological substances by neutron scattering and Mössbauer spectroscopy.33– 39

In these techniques, the mean-squared displacement (MSD), , of hydrogen atoms

experiencing vibrational motion is obtained. The results for numerous biological systems (proteins, peptides, membranes, RNA and DNA) show that motions of hydrogen atoms in these matrices change remarkably at 180–230 K.33-39 At lower temperatures, the MSD has linear temperature dependence, which is consistent with harmonic vibrational motion. At higher temperatures, however, the MSD temperature dependence becomes much steeper. For this reason, the vibrational motion in this temperature range is considered to be anharmonic or diffusive. The transition between these two motional regimes is called the dynamical transition. The temperature where starts to increase is called dynamical transition temperature, Td. In this work, we performed an ESE study of molecular motions in saturated phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers hydrated in the presence of either the sugar alcohol sorbitol or the disaccharides sucrose or trehalose. For brevity, all of these substances are simply called sugars for the remainder of this article.

Experimental DPPC lipids were obtained from Avanti Polar Lipids. Sucrose, trehalose and sorbitol were obtained from Sigma. Spin-labeled 5-DOXYL-stearic acid (5-DSA):

O

N

O OH

and 16-DOXYL-stearic acid (16-DSA): O OH N

O


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were obtained from Sigma-Aldrich. The stearic acids were dissolved in chloroform with 30 mg of DPPC in a molar ratio of 1:100. The solvent was removed by nitrogen gas flow followed by 24 h in storage under vacuum. The phospholipids were hydrated for 48 h at temperatures above (by ~ 5oC) the gel-to-fluid phase transition temperature (41oC), in pure water, a 0.2 M aqueous sugar solution or a 1 M aqueous sugar solution. The lipid-to-liquid ratio was ~1:1 by weight. The samples were frozen rapidly by immersion in liquid nitrogen. In some experiments, the small spin probe nitroxide 2,2,6,6-tetramethylpiperidone-Noxyl (TEMPONE), kindly donated as a gift from Dr. I. A. Kirilyuk, was used, which was dissolved in the aqueous 1 M sugar solutions in a concentration of 1 mM. A Bruker ELEXSYS E580 9-GHz FT-EPR spectrometer (Bruker, Germany) equipped with a dielectric resonator (Bruker ER 4118X-MD5) inside an Oxford Instruments CF 935 cryostat was used. In continuous wave EPR experiments, the incident microwave power was controlled optimized to ensure the absence of saturation of the EPR spectra. In pulse experiments, the cavity was overcoupled to provide ringing time ~ 100 ns. A two-pulse ESE sequence (16 ns pulse -  - 32 ns pulse - τ - echo) was used. The pulse amplitudes were adjusted to provide π/2 and π turning angles of the first and second pulses, respectively. To acquire ESE decay time traces, the time delay  was scanned with a step of 4 ns. All data treatment processing was performed on a PC using Origin software. The cryostat was cooled with flowing cold nitrogen gas. The sample temperature was controlled with an accuracy of 0.5 K.

Results CW EPR spectra The EPR spectrum of nitroxides consists of three hyperfine components corresponding to the three projections m of the nitrogen nuclear spin (I = 1) on its quantization axis (i.e., m = 0, 5 ACS Paragon Plus Environment


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±1). The EPR spectra of the systems under study are transforming slightly as the temperature increases (see Fig. 1S in Supporting Information). In particular, the separation between the two outmost peaks, which corresponds to twice the principal component, Azz, of the hyperfine tensor decreases with increasing temperature. The 2Azz values are collected in Fig. 1 for all three sugars, for concentration of 1 M, including the results for both 5-DSA and 16-DSA. Data are given in comparison with analogous data when the bilayer is hydrated by pure water.

Fig. 1. The temperature dependence of the splitting 2Azz between the two outmost peaks in CW EPR spectra for DPPC bilayers with incorporated 5-DSA (top) and 16-DSA (bottom) hydrated either in pure water or in presence of sugars. At low left corner the inaccuracy of experimental data is shown. (Online version in color.)


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One can see in Fig. 1 that the 2Azz value is higher for 5-PCSL than for 16-PCSL. This result can be readily explained in terms of a higher polarity of the surrounding in the former case.40 The 2Azz values begin to decrease above ~ 200 K and then decrease substantially above ~ 230 K. The 2Azz temperature dependencies appear to be similar for solvation by aqueous sugar solutions and by pure water.

Echo decays The major contribution to the echo decay in organic solids arises from the electron-nuclear spin interactions of the unpaired electron with surrounding protons in the matrix. These interactions fluctuate either because of spin diffusion in the proton subsystem or because of molecular motions of the neighboring atomic groups containing protons. Furthermore, static electron-nuclear interactions induce visible oscillations in the decay curves (electron spin echo envelope modulation, ESEEM).41,42 Because these contributions to echo decay are normally field-independent for nitroxides in organic solids, one may extract the pure contribution of orientational vibrations of the spin label. This is achieved by numerically dividing the time traces of the two field positions possessing different spectral anisotropy, assuming that the echo decay induced by the orientational motion of the spin label and the other contributions occur independently. The central component (m = 0) in the EPR spectrum of nitroxide is not influenced by the anisotropy of hyperfine interactions; therefore, it possesses the narrowest linewidth. The broadest high-field component (m = –1) is the most anisotropic. The insert to Fig. 2 shows the result of numerical division of the time traces obtained at the 1st and 2nd field positions. Note that the ESEEM practically disappears after this division, indicating that it is indeed field-independent. One can also see that the ratio of the echo decays is rather well approximated by the exponential function, with the rate of the exponential decay denoted as Wanis. I.e. the ratio of the two echoes decays as exp(-2τWanis). We estimated that experimental uncertainty in measurements of Wanis 7 ACS Paragon Plus Environment


consists of two components: the absolute one that is about ±0.05μs-1 and the relative one that is about ±0.04 Wanis .

2 1,0

5

1

-1

3

echo, a.u.

echo1/echo2

4

Wanis, s

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345 0,5

B, mT

350

echo2

echo1

2 0,0 0,2

0,4

0,6

, s

0,8

1,0

1

0

80

100

120

140

160

180

200

220

240

T, K

Fig. 2. Inset: An example of two time traces with time delay τ varied (adjusted to the same value at the beginning) obtained at field positions 1 and 2 and their ratio (noisy line) with its exponential approximation (smooth line). The exponential dependence determines the anisotropic relaxation rate, Wanis, which is given for different temperatures for DPPC bilayers prepared with spin-labeled 5-DSA (circles) or 16-DSA (squares). Open symbols: bilayers solvated by pure water; filled symbols: bilayers solvated by 1 M aqueous sorbitol solutions. The experimental inaccuracy linearly depends on Wanis (see text for details), and is shown for the limiting case of large Wanis; for small Wanis it coincides with the size of experimental points. (Online version in color.)


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5

4

Wanis, s-1

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3

2

1

0

80

100

120

140

160

180

200

220

240

T, K

Fig. 3. The same as in Fig. 2 but for sucrose solutions. (Online version in color.)

The obtained Wanis values are presented in Figs. 2 – 4 for all three sugars, for DSA labeled at the 5th or 16th carbon atom positions, for sugar concentrations of 1 M and 0 M (i.e. hydration by pure water). The data for the 0.2 M aqueous sugar solutions is given in the Supporting Information. The small negative values of Wanis observed at low temperatures can be readily ascribed to the phenomenon of instantaneous spectral diffusion in ESE of nitroxides, which results in a faster decay rate for the narrowest central component.24 This negative contribution depends on spin concentration, which could vary from sample to sample, because of slight uncertainty in sample preparations, and is likely to be the reason why the Wanis values for different samples differ slightly at low temperatures (80 K) – see Figs. 2 – 4. However, this effect is temperature-independent so it does not influence an analysis of the general trends of the Wanis temperature dependence.



As seen in Figs. 2 – 4, Wanis begins to sharply increase above 170 – 200 K. This behavior is very similar to observations made from neutron scattering and Mössbauer absorption experiments33–39 where the MSD of atoms is measured.

5

4

-1

3

Wanis, s

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2

1

0

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100

120

140

160

180

200

220

240

T, K

Fig. 4. The same as in Fig. 2 but for trehalose solutions. (Online version in color.)

Above 200 K, Wanis is slightly suppressed in the presence of sugars relative to the case of hydration by pure water. These observations are in agreement with previous data on DPPC bilayers hydrated in the presence of sucrose and sorbitol.23 (Note that in Ref 23 a different meaning of anisotropic relaxation rate was used which resulted in its twice larger value.) The presence of sucrose and trehalose influences the Wanis (Figs. 3 and 4) in a somewhat different way relative to sorbitol (Fig. 2); specifically, Wanis immediately begins to increase above ~ 120 K (in presence of trehalose this effect is observed only for 16th label position). The data within the Supporting Information show that changing the sugar concentration between 0.2 M and 1 M leads to a slight change of Wanis in the case of sorbitol. For sucrose and 10 ACS Paragon Plus Environment

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trehalose, the data for the 0.2 M aqueous sugar solution are close to the data of 1 M sugar solution. For comparative purposes, Wanis was also obtained for the spin probe TEMPONE in aqueous sugar solutions (Fig. 5). As it is seen in the insert of Fig. 5, the original data show that the contribution due to instantaneous diffusion is somewhat larger than that observed in Figs. 2 – 4. This may occur because of a slightly inhomogeneous distribution of the spin probes in the aqueous sugar solutions, which can result in their enhanced local concentration. To eliminate this unwanted contribution, the data in Fig. 5 are corrected by adding a temperature-independent value (2.0 µs-1 for sucrose and trehalose and 3.3 µs-1 for sorbitol solutions). Data on Wanis obtained for DPPC bilayers hydrated by pure water (cf. Figs. 2 – 4) are also presented in Fig. 5. Clearly, all the data sets agree closely with each other.

4

5 3 2 1 0

-1

3

Wanis, s-1

4

Wanis, s

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-1

2 -2 100

150

T, K

200

250

1

0

80

100

120

140

160

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T, K

Fig. 5. The Wanis obtained at different temperatures for spin probe TEMPONE in sorbitol (squares), sucrose (up triangles) and trehalose (down triangles) 1 M aqueous solutions, corrected 11 ACS Paragon Plus Environment


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for the instantaneous diffusion contribution (see text; insert shows original data). For comparison, the data for 5-DSA in DPPC bilayers hydrated by pure water are given (empty circles, the same data as in Figs. 2 – 4). (Online version in color.)

Discussion As shown previously in three-pulse stimulated echo experiments on 5-DSA and 16-DSA in DPPC bilayers at 200 K and 220 K,23 the Wanis value may be safely interpreted within the model of stochastic nanosecond molecular librations, at least at these temperatures. In line with this interpretation, the measured Wanis value is equal to the product R2c where is the mean-squared angular amplitude of motion, c is the correlation time, and R2 is a numerical coefficient equal approximately30 to ~ 1017 s-2, so that

Wanis ≈ (1017 s-2)c.

(1)

In this respect, Wanis data could be compared31,32 with the mean-squared displacement of atoms, , obtained from neutron scattering and Mössbauer absorption experiments.33-39 For different biological systems (membranes, peptides, proteins, DNA and RNA), was found to increase sharply just above ~ 200 K, which is what we observe herein for Wanis. Hence, the Wanis increase above 200 K may be related to dynamical transition phenomena discussed widely in the neutron scattering and Mössbauer absorption literature.33-39 The data in Fig. 5 may be compared with data from a neutron scattering study performed on analogous aqueous sugar solutions.43 The MSD determined from the neutron scattering experiments begin to increase above 230 K. This finding is inconsistent with the data in Fig. 5, where the Wanis increase is seen already above 170 K. This discrepancy could result from the different timescales of motion probed in the different techniques: ESE probes motions with correlation times up to ~ 100 ns, while neutron scattering is sensitive to a shorter ps to ns 12 ACS Paragon Plus Environment

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timescale. Consequently, in the ESE approach, more motional pathways could contribute to the experimental time window. CW EPR spectra are also sensitive to molecular librations and dynamical transition,44,45 because the measured 2AZZ splitting is an apparent one, diminished by motional averaging by 0 0 the value of 2 AZZ where AZZ is a “true” value, when the spin label is fully immobilized.

However, several disadvantages of the CW EPR approach make it less favorable than the ESE approach for detecting dynamical transition. First, CW spectra can be easily saturated at low temperatures, which results in their distortion. Second, CW spectra are noticeably influenced by harmonic motions.46,47 Third, except for molecular motion, the polarity of the environment can also strongly influence the spectral splitting.40 And finally, the precision of measurements in CW EPR could be much lower than that in ESE. Indeed, in CW EPR this precision is determined by inhomogeneous EPR line broadening in glassy media, which results in the uncertainty associated with AZZ to be approximately 0.1 – 0.2 G. This uncertainty allows one to estimate the smallest to be measured as ~ 5 × 10-3 0 rad2 (as AZZ is ~ 35 G). From the other side, the sensitivity of ESE experiments can be roughly

estimated in the following way: the ESE experimental uncertainty of obtaining Wanis is of the order ~ 105 s-1 (for the experimental points near 200 K). So, the lowest   2  value that can be measured with this technique is estimated with Eq. (1) as ~ 10-12/τc. In the most favorable case of

 c ~ 107 s (motions with τc > 10-6 s are too slow to be detected in two-pulse ESE and they are field-independent), the best estimate for the sensitivity of ESE experiments is ~ 10-5 rad2; which is almost three orders of magnitude smaller compared with the above estimation for CW EPR. The data presented in Fig. 1 show that CW EPR spectra are indeed temperaturedependent, and the 2Azz splitting decreases with temperature. However, a clear decrease of 2Azz begins at temperatures in the range of 240 – 250 K, where the Wanis obtained in the ESE 13 ACS Paragon Plus Environment


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experiments increases very rapidly and already attains rather high values (cf. Figs. 1 – 4). As mentioned above, this difference may occur because of the different sensitivities of the two approaches to stochastic molecular librations. Also, the possible influence of increasing polarity with temperature on the Azz may take place. Note that the and c values cannot be obtained separately from ESE experiments, and this is a disadvantage of the spin-label ESE approach. However, as coherent harmonic motion does not produce spin relaxation, the onset of dynamical transition in ESE decays could be observed more clearly than in neutron scattering or Mössbauer absorption. The data for the influence of sucrose and trehalose on the Wanis values in DPPC bilayers (Figs. 3 and 4) show that Wanis begins to increase at 120 K (for the case of trehalose this effect is observed only for the 16th label position). Analogous behavior has been observed previously for two other spin-labeled bilayers – for cholesterol-containing DPPC bilayers, when the lipids are spin-labeled at the 16th carbon atom position along the acyl chain,25 and for bilayers prepared from unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids.48 For both of these systems, additional freedom for motion occurs because of increased flexibility of the lipid structure beyond the cholesterol nucleus in the former case, and because of disorder induced by the kink in the POPC structure in the latter case. Accordingly, we suggest that sucrose and trehalose interact with the membrane surface and increases the inter-lipid spacing (even at a small sugar concentration of 0.2 M – see data in Supporting Information). This conclusion is in agreement with numerous other data available in the literature.5-11,19,49,50 As the effect is more pronounced for sucrose solution, one may suggest that sucrose interacts more strongly with the membrane than trehalose does (cf. Figs. 3 and 4). As it was mentioned above, Wanis is slightly suppressed above 200 K in the presence of sugars relative to the case of hydration by pure water. This is most pronounced for solvation by aqueous sorbitol solution (see Fig. 2) where this suppression is seen already above 170 K. This suppression looks like shifting the dynamical transition temperature Td from ~ 170 K when 14 ACS Paragon Plus Environment

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bilayer is solvated by pure water to ~ 200 K when bilayer is solvated by aqueous sorbitol solution. It may be explained taking into account that sorbitol probably can penetrate into DPPC bilayer – the effect directly observed for glycerol molecules using ESEEM approach,51 which are only twice smaller than sorbitol molecules. (Note that data in Fig. 1 show that the AZZ values slightly increase at low temperatures in presence of sorbitol which also may be explained by penetration of the polar sorbitol molecules.) The presence of sorbitol in the bilayer can increase local microviscosity in the lipid surrounding thus suppressing molecular motions. Note that this influence of sorbitol depends on its concentration – see Fig. 2S in Supporting information. It is well-known that the phase transition from liquid water to ice damages cells and certainly leads to their death. The presence of molecular motions in aqueous sugar solution is obvious evidence that ice is not formed in the nearest surrounding of guest molecules in these solutions. Moreover, the data in Fig. 5 show that motions in aqueous sugar solutions are similar to motions in DPPC bilayer. So the motional characteristics of lipids in the membrane at low temperatures correlate with those in the sugar aqueous solutions. By other words, sugar solutions mimic bilayer, in the sense of motional characteristics of molecules. Therefore lipid molecules are not expected to experience upon freezing the severe change of physical properties of the surrounding liquid, as it must be when ice is formed. Note finally that stearic acids are known to exist in two forms – ionic and neutral which for 5-DSA at room temperature even could result in different EPR spectra.52 One may suppose that the ionic form is anchored tightly by the charge at the membrane surface while the neutral one is sinking deeper into the bilayer. Indeed, the NMR data53 indicate that the spin label groups on the stearates are located nearer to the membrane exterior than the analogous positions of the unlabeled phospholipid acyl chains. However as in the above discussion the exact label positions were not employed (data for the 5-DSA and 16-DSA were qualitatively related only to the positions closer to the surface and those in the middle of the bilayer, respectively), the



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existence of two forms might not induce a problem in the application of 5-DSA and 16-DSA in our case.

Conclusions From a methodological point of view, the present results show that spin-echo EPR of spin labels can provide useful information on the molecular mechanisms of action of cryoprotective agents of biological systems. An obvious advantage of this approach is the possibility to study molecular motions at low temperatures at which the system is frozen. ESE signal decay data can be interpreted within the model of stochastic nanosecond molecular librations. ESE decays result in measurement of the anisotropic relaxation rate, Wanis, which is directly connected with motional characteristics by the Eq. (1), under the present experimental design. These stochastic librations in biological media most likely originate from the same source as the anharmonic or diffusive motions of atoms, as detected by neutron scattering or Mössbauer absorption spectroscopy. For DPPC bilayers hydrated in the presence of sucrose or trehalose, evidences were found for the direct interaction between the sugar and bilayer. However this interaction was not observed in the case of hydration in the presence of sorbitol. Probably, for cryoprotective action important could be the found similarity of the low-temperature molecular motions in the bilayer and those in the aqueous sugar solutions. So, cryoprotection of living cells may be provided by the similarity of molecular motions in the cellular membranes and in the surrounding cryoprotectant-containing intercellular liquid. Then biomolecules molecules upon freezing are not expected to experience the severe change of physical properties of the surrounding liquid, as it must be when the intercellular ice is formed.


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Acknowledgments This work was supported by RFBR Grant # 12-03-00192.

Supporting Information Available: Representative CW EPR spectra for DPPC bilayers with incorporates spin-labeled stearic acids and Wanis temperature dependence for different sugar concentrations (0 M, 0.2 M and 1 M). This material is available free of charge via the Internet at http://pubs.acs.org.



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4 relaxation rate, s-1

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Spin relaxation induced by molecular motions of spin labels in lipid bilayer is similar to that in aqueous sugar solutions

Sorbitol solution Trehalose solution Sucrose solution DPPC bilayer

2

0 100

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T, K

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Low-Temperature Molecular Motions in Lipid Bilayers in the Presence

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