Heterogeneities in Cholesterol-Containing Model Membranes Observed by Pulsed Electron Paramagnetic Resonance of Spin Labels.

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Heterogeneities in Cholesterol-Containing Model Membranes Observed by Pulsed EPR of Spin Labels Maria E. Kardash, Nikolay P. Isaev, and Sergei Andreevich Dzuba J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b03080 • Publication Date (Web): 12 May 2015 Downloaded from http://pubs.acs.org on May 16, 2015

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The Journal of Physical Chemistry

Heterogeneities in Cholesterol-Containing Model Membranes Observed by Pulsed EPR of Spin Labels

Maria E. Kardash, Nikolay P. Isaev and Sergei A. Dzuba*

Institute of Chemical Kinetics and Combustion, 630090 Novosibirsk, Russia, and Novosibirsk State University, 630090, Novosibirsk, Russia

*Corresponding author. Fax: +7(383) 330 7350, e-mail address: [email protected]

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Abstract Biological membranes are supposed to have heterogeneous structure containing lipid rafts – lateral micro- and nano-domains enriched in cholesterol (chol) and sphingolipids. In this work, lipid bilayers containing a small amount of the spin-labeled chol analog 3β-doxyl-5α-cholestane (chlstn) were studied using electron spin echo (ESE) spectroscopy, which is a pulsed version of electron paramagnetic resonance (EPR). Bilayers were prepared from an equimolecular mixture of

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

(DPPC)

and

1,2-dioleoyl-sn-glycero-3-

phosphocholine (DOPC) with chol added at different concentrations. The ESE decays recorded at 77 K become faster with increase of chlstn concentration. The chlstn-dependent contribution to ESE decay is remarkably non-exponential; however, the logarithm of this contribution can be rescaled for different chlstn concentrations to a universal function with the rescaling factor approximately proportional to concentration. This result shows that the chlstn-dependent contribution to the ESE decay can be employed to estimate the local (at the nanometer scale of distances) chlstn concentration. Analogous rescaling behavior is also observed for the bilayers with different chol concentrations, with the rescaling factor increasing with increase of the chol concentration. This result is evidence that chlstn molecules are distributed heterogeneously in the chol-containing bilayer and form clusters with enhanced chlstn (and probably chol) local concentration. The local concentration of chlstn molecules for large chol content (~ 30 mol. %) was enhanced by at least ~70 % versus chol-free bilayers. The suggested approach appears to be useful for exploring heterogeneities in lipid composition of biological membranes of different types.

Keywords: biological membranes, lipid rafts, EPR, ESE, nanoclusters, instantaneous diffusion in electron spin echo


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Introduction There is increasing evidence for the compositional heterogeneous structure of plasma membranes that are assumed to contain lipid rafts – fluctuating nanoscale assemblies of lipids, cholesterol (chol), and proteins.1-5 Lipid rafts are thought to form platforms that function in membrane signaling and trafficking. The properties of lipid rafts have been extensively studied with a variety of biophysical and biochemical techniques including detergent extraction,6 electron microscopy,7 fluorescence microscopy,8 near-field scanning optical microscopy,9 fluorescence resonance energy transfer microscopy,10 fluorescence correlation spectroscopy,11 single-molecule spectroscopy,12 spin-label electron paramagnetic resonance (EPR),3,13 computer simulations,14 etc. The results support the hypothesis that lipid rafts are small, dynamic domains in membranes enriched in cholesterol, saturated lipids (sphingomyelin or others), and membrane proteins. However, their small size (10-100 nm) is below the resolution limit of optical microscopy. Thus, there is no acceptable agreement in the literature regarding the properties of lipid rafts.2,4,15 Thus, new experimental tools are urgently needed to characterize these structures. We suggest here that a spin-labeled EPR approach based on pulsed EPR spectroscopy,16 in a version of electron spin echo (ESE), could be used to detect the formation of heterogeneities in the lipid bilayers. Decay of the ESE signal depends on magnetic dipole-dipolar interactions between unpaired electrons of the spin labels. The mechanisms of this dependence17,18 are determined by stochastic fluctuations of these interactions that are induced by electron spinlattice relaxation and/or mutual electron spin flips as well as by sudden modulation of these interactions when applying the echo-forming microwave pulses (a so-called “instantaneous diffusion” mechanism19). The ESE technique is a promising tool for researching nanoscopic spatial inhomogeneities in biological membranes because these mechanisms are sensitive to the interactions between unpaired electrons of spin labels at the nanoscale. The bilayers used here were prepared from an equimolecular mixture of 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 3 ACS Paragon Plus Environment


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with chol added at different concentrations. This lipid composition is often used as a model for the outer leaflet of the animal cell plasma membrane.20 We used a chol spin-labeled analog, 3βdoxyl-5α-cholestane (chlstn), for EPR applications. Bilayers were prepared as multilamellar vesicles (MLV).

Experimental The phosphatidylcholines DPPC and DOPC and spin-labeled cholesterol analog chlstn were obtained from Avanti Polar Lipids (Birmingham, AL). The chemical structures of chol and chlstn are given below:

cholesterol (chol)

3β-Doxyl-5α-cholestane (chlstn)

The lipids DPPC and DOPC were co-dissolved in chloroform at 1:1 w/w with chlstn added at three different concentrations of 0.5, 1 and 2 mol.%. The accuracy of the chlstn concentration was better than ±0.2 mol.%. The solvent was removed by nitrogen flow followed by storage for 12 h under vacuum (10-2 bar). The resulting samples were hydrated for 2 h at room temperature by adding doubly distilled water at a lipid/water ratio of 1:4 w/w. The samples were put into 3-mm outer diameter glass tubes and frozen by immersion in liquid nitrogen. A Bruker ELEXSYS E580 9-GHz FT-EPR spectrometer (Bruker, Germany) was used and equipped with either a split-ring resonator (Bruker ER 4118X-MS3) or a dielectric resonator (Bruker ER 4118X-MD5) inside an Oxford Instruments CF 935 cryostat. The split-ring resonator was used in the pulsed experiments while the dielectric resonator was used in continuous wave (CW) experiments. The incident microwave power in the CW experiments was controlled to 4 ACS Paragon Plus Environment

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ensure the absence of saturation in the EPR spectra. In pulse experiments, the resonator was overcoupled to provide a short ring time (~120 ns). A two-pulse ESE sequence was employed (16-ns pulse - τ - 32-ns pulse - τ - echo). The pulse amplitudes were adjusted to provide turning angles of π/2 and π for the first and second pulses, respectively. To acquire ESE decays, the time delay τ was scanned, with a 4 ns step. All data processing was performed on a PC. The cryostat was cooled by flowing cold nitrogen gas. The sample temperature was controlled to ±0.5 K.

Results and Discussion Conventional CW EPR spectra of spin-labeled chlstn molecules in DPPC/DOPC bilayers were recorded at 200 K. This temperature is not so high that the EPR spectra would remain immobilized (i.e. motional effects are frozen out), but not so low that saturation effects in EPR become important. Representative EPR spectra are shown in Fig. 1 for 2 mol.% chlstn content and different chol concentrations. Fig. 1 shows that the absence of chol significantly broadens the EPR line. This broadening can be readily explained by considering the long-range lipidmediated orientational self-ordering of chlstn molecules in domains in the bilayer previously described.21 Indeed, equal orientations of chlstn molecules implies single-crystal-like narrow EPR lines in the domains which can result in an intensive spin-exchange process22 that is known to induce a substantial EPR line broadening. In the presence of chol, the ordering could be destroyed because chol molecules contain a hydroxyl group in its structure that strongly interacts with the lipid heads to form hydrogen bonds23 such that their orientation in the membrane is determined mainly by this interaction. This destruction of order would certainly suppress spin exchange and make the total line narrower. Data in Fig. 1 show that 2 mol. % of chol is enough to destroy ordering of 2 mol. % of chlstn molecules. All ESE measurements were performed at 77 K. Fig. 2 presents a semilogarithmic plot of the selected ESE decays collected at the maximum of EPR absorption line for DPPC/DOPC/chol 5 ACS Paragon Plus Environment


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bilayers with chlstn added at different concentrations. The small oscillations seen on the decay curves are induced by static electron-nuclear interactions with neighboring proton spins. This effect is the electron spin echo envelope modulation (ESEEM).16 Fig. 2 illustrates that the ESE decays become faster with increasing either the chlstn or chol concentration. Molecular motions are suppressed at low temperatures, and the ESE decays are determined by “instantaneous diffusion” mechanism and by relaxations induced by the interaction of unpaired electron of spin label with fluctuating spins of the nearby nuclei as well as by fluctuating and static interactions with other electron spins17,18. The electron-nuclear interactions fluctuate mostly due to spin diffusion in the proton environment; relaxation by electron-electron interactions is induced by fluctuation of electron spins (spin-lattice relaxation, spin diffusion). The ESE decays in Fig. 2 for chlstn at 0.5 mol.% are induced mostly by electronnuclear interactions while for chlstn at 1 mol.% and 2 mol.% the electron-electron interactions also become important. The contribution of the electron-electron mechanism to ESE decay can be derived by subtracting the data presented in a semilogarithmic scale (Fig. 2) for different chlstn concentrations because the electron-nuclear contribution is expected to be eliminated after this subtraction. Fig. 3a shows the results of the subtraction in which the 0.5 mol.% chlstn sample was used as a reference. One can see in Fig. 3a that the presence of chol remarkably enhances the chlstn-concentration contribution to the ESE decay. In Fig. 3b the ESE decays given in Fig. 3a are rescaled to show that, first, the data for different chlstn concentrations and the same chol content practically coincide when being multiplied by some numerical factor Fchlstn. Second, data for the same chlstn concentration and different chol content can also be rescaled by introducing a rescaling factor Fchol. From the first statement, the empirical relationship follows

2 = exp − 2,

(1) 6

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where Cchlstn is chlstn concentration, and 2 is some universal function. The rescaling factor

Fchlstn was close to the ratio of the concentrations for samples under comparison (see caption to Fig. 3b) when considering the experimental uncertainty of determining chlstn concentration (see Experimental). This means that the experimentally obtained chlstn-contribution to the ESE decay can be used for assessment of chlstn local concentrations. The term “local” means that spin-spin interactions between electron spins influence echo decays when spins are separated on the nanometer scale. Local concentration may differ from the mean if the spatial distribution of the spin labels is heterogeneous. The second statement above implies that Eq. (1) remains valid when comparing samples with the same chlstn concentration and different chol content. The rescaling factor Fchol reflects changes in the local chlstn concentration when the two samples with different chol contents are compared. The capacity for rescaling, analogous to that shown in Fig. 3b, was also found for DPPC/DOPC/chol bilayers with other chol concentrations (data not given). The rescaling factor

Fchol obtained in these measurements, is given in Fig. 4 as a function of chol content. One can see that Fchol increases with chol content and reaches a value of Fchol ≈ 2.5. This implies that the bilayer becomes heterogeneous in the presence of chol. As Fchol > 1, the Fchol may be considered to be an enhancing factor. Two straight lines drawn in Fig. 4 clearly demonstrate that the concentration dependence consists of two distinct regions. There is a fast increase below 2 mol.% chol that is most likely related to the mentioned above destruction of the orientational chlstn domains. This explanation is supported by the narrowing of the EPR seen in Fig. 1, which also occurs near the 2 mol.% of chol. Further increases in Fchol could be assigned to the increasing local concentration of chlstn in the bilayer. This implies the appearance of heterogeneity in the bilayer structure with increasing chol content. For chol content varying from 2 mol.% to 29 mol.%, the enhancing factor Fchol 7 ACS Paragon Plus Environment


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increases from 1.5 to 2.5 (see Fig. 4), i.e. almost by 70 %. This seems to be a reliable lowest value of concentration enhancement in chol-containing samples. Regarding raft formation, one would expect a discontinuous behavior for the enhancing factor Fchol when phase transition accompanies raft formation.1-15 This behavior is not observed in our study (see Fig. 4), except for the sudden drop of the slope near at chol 2 mol.% that could be explained by destruction of ordered domains of chlstn molecules – see above. Probably the observed continuous behavior of the enhancing Fchol factor implies that in the gel phase the raft formation occurs in a stretched way when chol concentration is varied. However, this issue needs further investigation. The data in Fig. 3 show that the subtracted decays are non-linear in the semilogarithmic plot, i.e. the function 2 is non-linear. Note that this non-linearity may appear because of the two-dimensional nature of electron-electron interactions for which the logarithm of ESE decay is

expected to be proportional to .24 In our case however, the ESE decays remain nonlinear even

if the data in Fig. 3 are re-plotted against (result is not shown). Thus, to explain the nonlinearity of 2, one may again admit a non-homogeneous spatial spin distribution even in the absence of chol. This is not surprising because the chemical structures of chlstn and chol are very similar, and 2 mol.% of chlstn can result in similar structural effects in the membrane as 2 mol.% of chol. The analogous non-linearity of ESE decays was observed in numerical applications of pulsed electron-electron double resonance (PELDOR).24-26 This technique is based on instantaneous diffusion in ESE decays similar to that in background of the two-pulse ESE experiment. Note that enhancement of local chlstn concentration with increasing chol content does not manifest itself in conventional EPR spectra for large chol content (see Fig. 2). This could have two explanations. First, simple estimation shows that 70 % increase of 2 mol. % concentration of spin label would result in EPR line dipolar broadening by about ~ 0.01 mT, which is below spectral resolution in our case. And second, with increase of chol content the 8 ACS Paragon Plus Environment

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EPR linewidth is expected to decrease because of lowering of proton density in the nearest spin label surrounding – the effect which acts in the reverse direction to the expected dipolar line broadening. From the other side, the advantage of ESE technique is its high sensitivity to electronelectron dipolar interactions, because of lifting all static sources of EPR line broadening. And, in the approach suggested here, ESE decay is free from influence of electron-nuclear spin-spin interactions. Note however that conventional CW EPR spectra provide important information in our case that chlstn molecules do not aggregate in the membrane; otherwise one would expect a severe distortion of EPR lineshape in Fig. 1 due to spin exchange process.22

Conclusions Here, we used a new ESE-based approach to observe heterogeneities in membrane structures. The data for chlstn–the spin-labeled chol analog–in DPPC/DOPC/chol bilayers show remarkable dependence of ESE decays on chol concentration, which suggests that domains are formed with enhanced chlstn concentration in the presence of chol. The observed enhancement is in general agreement with the concept of cholesterol-lipid raft formation in biological membranes. The enhancement of the local concentration in this study approaches 70%. The observed effect may imply that the chol local concentration is similarly increased in these domains because the molecular structures of chol and chlstn are very similar. This approach may be used to study spatial heterogeneities (lipid rafts) in different types of biological membranes including plasma membranes of living cells. Of course it is necessary to employ low temperatures in these studies; otherwise, motion effects would suppress the ESE signal. However, the results obtained at low temperatures could provide a clue to understanding important properties of membrane heterogeneities at physiological temperatures as well.



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The data also clearly show that the previously found long-range orientational selfordering of chlstn molecules in the bilayer21 is destroyed in presence of chol. Most likely that this is because chol molecules contain a hydroxyl group that strongly interacts with lipid heads via formation of hydrogen bonds. Thus, chol orientation in the membrane is determined mainly by this strong interaction that destroys the self-ordering orientation induced by weaker lipidmediated long-range interactions21 of chlstn molecules in biological membranes.

Acknowledgments This work was supported by RFBR Grant # 15-03-02186.


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Biophys. Biomol. Struct. 2004, 33,269–295. 2. Lingwood, D.; Simons, K. Lipid Rafts as a Membrane-Organizing Principle. Science. 2010, 327, 46–50. 3. Ionova,

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of

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Microdomains in Intact Cell Membranes. Annual. Rev. Phys. Chem. 2005, 56, 309-336. 8. Nichols, B. J.; Kenworthy, A.K.; Lippincott-Schwartz, J. Rapid Cycling of Lipid Raft Markers between the Cell Surface and Golgi Complex. J. Cell Biol. 2001, 153, 529–541. 9. van Zanten, T.S.; Cambi, A.; Koopman, M.; Joosten, B.; Figdor, C.G.; Garcia-Parajo, M.F. Hotspots of GPI-Anchored Proteins and Integrin Nanoclusters Function as Nucleation Sites for Cell Adhesion. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 1855718562. 10. Goswami, D.; Gowrishankar, K.; Bilgrami, S.; Ghosh, S.; Raghupathy, R.; Chadda, R.; Vishwakarma, R.; Rao, M.; Mayor, S. Nanoclusters of GPI-Anchored Proteins are Formed by Cortical Actin-Driven Activity. Cell 2008, 135, 1085-1097. 11 ACS Paragon Plus Environment


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Phys. Rev. 1962, 125, 912-932. 20. Mills, T.T.; Tristram-Nagle, S.; Heberle, F.A.; Morales, N.F.; Zhao, J.; Wu, J.; Toombes, G.E.S.; Nagle, J.F.; Feigenson, G.W. Liquid-Liquid Domains in Bilayers Detected by Wide Angle X-Ray Scattering. Biophys. J. 2008, 95, 682-690. 12 ACS Paragon Plus Environment

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Figure captions Fig. 1. The CW EPR spectra obtained at 200 K for 2 mol% chlstn in the DPPC/DOPC bilayer for different chol concentrations. For convenience of comparison, the spectra are adjusted to the same maximal intensity of the low-field shoulder.

Fig. 2. Semilogarithmic plot of the representative ESE decays taken at the maximum of the EPR spectrum. Samples are spin-labeled chlstn dissolved at different concentrations in the DPPC/DOPC/chol bilayers in the absence or presence of chol (23 mol.%). The curves in the latter case are shifted down along the vertical axis for convenience of visualization.

Fig. 3. The differences in the semilogarithmic scale of the original ESE decays given in Fig. 2 in which the reference E0(2τ) decay is taken for the 0.5 mol.% chlstn concentration. The curves are shifted along the vertical axis for convenience of visualization. (a) The E(2τ) decays refer to 1 mol.% and 2 mol.% chlstn in curves 1 and 2, respectively, for the chol content indicated in Figure. (b) Rescaling of curves given in (a) by multiplication by a factor Fchlstn or Fchol, where subscript denotes whether curves are compared to different chlstn and the same chol content, or vise versa, respectively. Top: for [chol] = 0, curve 1 is rescaled with Fchlstn = 2.1 to match curve 2. Middle: for [chol] = 23 mol. %, curve 1 is rescaled with Fchlstn = 2.5 to match curve 2. Bottom: curve 2 for [chol] = 0 is rescaled with Fchol = 1.89 to match the curve 2 for [chol] = 23 %.

Fig. 4. The rescaling (enhancing) factor Fchol obtained by adjusting the different curves as shown in Fig. 3b (bottom) for the DPPC/DOPC/chol bilayers with different chol content. The lines are drawn to guide the eye.


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[chol] (mol. %) 0 2 13 20 29

335

340

345

350

355

field, mTesla

Fig. 1. The CW EPR spectra obtained at 200 K for 2 mol.% chlstn in the DPPC/DOPC bilayer for different chol concentrations. For convenience of comparison, the spectra are adjusted to the same maximal intensity of the low-field shoulder.



0 [chol] = 0 [chlstn]

-2 [chol] = 23 mol. %

ln(E(2τ)/E(0.24 µs))

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(mol. %) 0.5 %

-4

1% 2%

-6

0.5 % 1%

-8

2%

-10

0

1

2

3

4

5

2τ, µs

Fig. 2. Semilogarithmic plot of the representative ESE decays taken at the maximum of the EPR spectrum. Samples are spin-labeled chlstn dissolved at different concentrations in the DPPC/DOPC/chol bilayers in the absence or presence of chol (23 mol.%). The curves in the latter case are shifted down along the vertical axis for convenience of visualization.


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0

[chol] = 0

a

1

ln(E(2τ)/E0(2τ))

2

-2 [chol] = 23% 1

-4

2

0

1

2

3

4

5

2τ, µs

Fchlstn ln(E(2τ)/E0(2τ)), Fchol ln(E(2τ)/E0(2τ))

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


0

b

[chol] = 0

-2 [chol] = 23 mol. %

-4

-6 [chol] = 0 and 23 mol. %

0

1

2

3

4

5

2τ, µs

Fig. 3. The differences in the semilogarithmic scale of the original ESE decays given in Fig. 2 in which the reference E0(2τ) decay is taken for the 0.5 mol.% chlstn concentration. The curves are shifted along the vertical axis for convenience of visualization. (a) The E(2τ) decays refer to 1 mol.% and 2 mol.% chlstn in curves 1 and 2, respectively, for the chol content indicated in Figure. (b) Rescaling of curves given in (a) by multiplication by a factor Fchlstn or Fchol, where subscript denotes whether curves are compared to different chlstn and the same chol content, or vise versa, respectively. Top: for [chol] = 0, curve 1 is rescaled with Fchlstn = 2.1 to match curve 2. Middle: for [chol] = 23 mol. %, curve 1 is rescaled with Fchlstn = 2.5 to match curve 2. Bottom: curve 2 for [chol] = 0 is rescaled with Fchol = 1.89 to match the curve 2 for [chol] = 23 %.



2,5

2,0

1,5

Fchol

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1,0

0,5

0,0 0

5

10

15

20

25

30

[chol], mol. %

Fig. 4. The rescaling (enhancing) factor Fchol obtained by adjusting the different curves as shown in Fig. 3b (bottom) for the DPPC/DOPC/chol bilayers with different chol content. The lines are drawn to guide the eye.


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TOC graphic image


Mapping of collision frequencies for stearic acid spin labels by saturation-recovery electron paramagnetic resonance.

Microscopic versus macroscopic diffusion in model membranes by electron spin resonance spectral-spatial imaging.

Integrated Computational Approach to the Electron Paramagnetic Resonance Characterization of Rigid 310-Helical Peptides with TOAC Nitroxide Spin Labels.

Assignment of a ligand in stellacyanin by a pulsed electron paramagnetic resonance method.

Reappraisal of the electron spin resonance spectra of maleimide and iodoacetamide spin labels in erythrocyte ghosts.

Selective placement of electron spin resonance spin labels: new structural methods for peptides and proteins.

Interactions of retinoids with phospholipid membranes: electron spin resonance.

Advanced paramagnetic resonance spectroscopies of iron-sulfur proteins: Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM).

Determination of molybdenum in seawater by electron paramagnetic resonance spectrometry.

Conformational change of spin labelled myoglobin. Electron paramagnetic resonance in solution and powdered samples.

Design and implementation of an FPGA-based timing pulse programmer for pulsed-electron paramagnetic resonance applications.

Electron spin resonance microscopy of an in vitro tumour model.

Coherence Transfer by Passage Pulses in Electron Paramagnetic Resonance Spectroscopy.

Electron spin resonance analysis of irreversible changes induced by calcium perturbation of erythrocyte membranes.

Electron spin resonance study of changes during development of solid yoshida tumour. II. Paramagnetic metal ions.

HtrII in membranes and nanodiscs.

Pulsed electron-electron double resonance spectroscopy between a high-spin Mn(2+) ion and a nitroxide spin label.

Electron paramagnetic resonance studies of carotenoids.

Probing the Hydrogen Bonding of the Ferrous-NO Heme Center of nNOS by Pulsed Electron Paramagnetic Resonance.

A genetically encoded spin label for electron paramagnetic resonance distance measurements.

Field-stepped direct detection electron paramagnetic resonance.

Electron spin resonance study on the permeability of superoxide radicals in lipid bilayers and biological membranes.

Electron paramagnetic resonance spectra of myeloperoxidase in polymorphonuclear leukocytes.

An electron paramagnetic resonance study of free radicals in cells.