MAGNETIC RESONANCE IN CHEMISTRYMagn. Reson. Chem. 2005; 43: S20–S25Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1680

High-field spin-label EPR of lipid membranes†

Derek Marsh,1∗ Dieter Kurad1 and Vsevolod A. Livshits2

1 Max-Planck Institut fur biophysikalische Chemie, 37070 Gottingen, Germany2 Centre of Photochemistry, Russian Academy of Sciences, Moscow, Russian Federation

Received 1 April 2005; Revised 23 June 2005; Accepted 23 June 2005

High-field EPR of spin-labelled lipid chains has proved to be an extremely productive means for biophysicalinvestigations of phospholipid bilayer membranes. Results on the following three topics are reviewed:

1. Non-axial ordering of lipid chains in cholesterol-containing membranes;2. Transmembrane polarity profiles and water penetration in lipid membranes;3. Role of HF-EPR in multi-frequency spectral simulations to investigate lipid-chain dynamics in

membranes.

These results are obtained from phosphatidylcholine spin probes with the nitroxide systematicallystepped down the sn-2 chain, in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine(DMPC)–containing cholesterol. Copyright 2005 John Wiley & Sons, Ltd.

KEYWORDS: EPR; ESR; spin label; nitroxide; membrane; phospholipid; bilayer; phosphatidylcholine; cholesterol

INTRODUCTION

Among other distinctive features of high-field EPR,1,2 threehave proved to be particularly decisive for applications to thestudy of spin-labelled lipids in biological membranes. Thefirst of these is the pronounced non-axial Zeeman anisotropyof nitroxyl spin labels at operating frequencies of 94 GHzand higher. The (gxx � gyy) anisotropy allows investigation ofthe lateral ordering of the lipid chains that could be relevantto the formation of in-plane membrane domains that arespecifically enriched in cholesterol.3,4 The second relevantfeature is the preferential sensitivity of the nitroxide gxx-tensor element to polarity, especially to hydrogen bondingwith water. In addition to mapping out the transmembranepolarity profile with spin-labelled lipids, the orientationalselection of nitroxide EPR at 94 GHz allows resolution ofthe lateral heterogeneity in water distribution at variousdepths in the membrane.5 The third decisive feature is theshift in the time window of motional sensitivity of nitroxideEPR to shorter correlation times, at higher Zeeman fields.This means that lipid spin-label EPR spectra at 94 GHz andabove are sensitive primarily to rapid internal motions; theslower overall rotations of the entire lipid molecule aredriven towards the rigid limit, even in the fluid membranephase.6,7 Results obtained with spin-labelled lipid chains inphospholipid bilayer membranes, in each of these three areasof high-field EPR, are reviewed here.

†Presented as part of a special issue on High-field EPR in Biology,Chemistry and Physics.ŁCorrespondence to: Derek Marsh, Max-Planck-Institut furbiophysikalische Chemie, Abt. Spektroskopie, 37070 Gottingen,Germany. E-mail: dmarsh@gwdg.deContract/grant sponsor: Deutsche Forschungsgemeinschaft;Contract/grant number: SPP 1051.

RESULTS AND DISCUSSION

Lateral ordering of lipid chains by cholesterolIn-plane membrane domain formation by lateral order-ing of phospholipid chains is indicated schematically inFig. 1(b). This implies a non-vanishing gxx � gyy anisotropyof nitroxide-labelled lipid chains in the xy plane. Any descrip-tion of the lateral ordering of the spin-labelled lipids in termsof the g-value anisotropy at high field, however, must alsotake into account the transverse ordering that corresponds toangular fluctuations about the membrane director (Fig. 1(a)).The g-tensor anisotropy is defined by:

g D gzz � 12 �gxx C gyy� �1�

for the axial anisotropy, and

υg D 12 �gxx � gyy� �2�

for the azimuthal, non-axial anisotropy. Similar relationshold for the 14N-hyperfine tensor, A, where υA D 0 fordipolar hyperfine interactions.

In the Redfield limit, the resonance positions in themotionally averaged spectrum are characterised by reducedg- and A-tensor anisotropies that are given by:4

hgi D hP2�cos �A�ig �3�

hυgi D 16 [2 C 3hcos �Ai C hP2�cos �A�i]hcos 2�� � ��iυg �4�

and

hAi D hP2�cos �A�iA �5�

where �A, � and � are defined in Fig. 1(a), and angularbrackets represent time averages. For rapid motion, which is

Copyright 2005 John Wiley & Sons, Ltd.

High-field spin-label EPR of lipid membranes S21

y

x−

N (membrane)ZA (spin)

H (field)

b

qA

XA

XA

−f−f

(a) (b)

Figure 1. (a) Relations between the instantaneous spin-labelz-axis (zA), director axis (i.e. membrane normal, N) and thelaboratory magnetic field direction, H. The nitroxide z-axis isassumed to coincide with the molecular long axis that hasuniaxial ordering, relative to the director, N. zA is inclined at �A

to N; the maximum amplitude of �A is ˇ. xA is the direction ofthe mean nitroxide x-axis which performs �-rotations about zA,with mean value � about which the maximum amplitude is� � � D �o. (b) Schematic arrangement of the lateral orderingof the chains in diacyl phospholipids. View is along the chainz-direction; upper part corresponds to complete lateralalignment within a domain (i.e. �o ³ 0°) and the lower partcorresponds to random lateral disorder (i.e. �o ³ 90°).

the case for the internal rotations at high field,7 the hyperfineanisotropy therefore depends only on the conventional orderparameter hP2�cos �A�i (in so far as υA ³ 0). The non-axial gxx � gyy anisotropy, however, depends additionally onhcos �Ai and hcos 2�� � ��i, as seen from Eqn (4). Therefore,to obtain the ‘order parameter’ hcos 2�� � ��i associated withthe azimuthal rotation, a value is required for hcos �Ai. Thiscan be obtained if the orientation pseudopotential U��A�governing the off-axis motion is known.

The simplest model for fluctuations in orientation of thenitroxide zA-axis is a restricted random walk within a coneof half-angle, ˇ (Fig. 1(a)). Each orientation is weighted bysin �A, and the z-axis order parameter is given by:

hP2�cos �A�i D 12 cos ˇ�1 C cos ˇ� �6�

where, ˇ maybe obtained from Eqn (5) by using theexperimental value of hAi. Then the angular averagerequired to interpret the non-axial g-value anisotropy isgiven in this model by:

hcos �Ai D 12 �1 C cos ˇ�. �7�

A corresponding model for the azimuthal xA-axis orderingis completely random �-angle fluctuations of maximumamplitude š�o about the mean value, �, then:

hcos 2�� � ��i D sin �o cos �o/�o �8�

In this simplified approach, the restricted angular motionis characterised completely by the independent maximumamplitudes, ˇ of the z-axis tilt, and �o of the rotation or twistof the x-axis about the z-axis (Fig. 1(a)).

Figure 2 shows the dependence on position, n, of chainlabelling of the lateral and transverse order parameters,

hcos 2�� � ��i and hP2�cos �A�i, for membranes of dimyristoylphosphatidylcholine (DMPC) C 40 mol% cholesterol in theliquid-ordered phase at 30 °C. The fast motional limit isassumed in deriving these values, because the slow overallmotion of the lipid molecule hardly affects the high-fieldspectra at 94 GHz (discussed later and in Ref. 7). Both orderparameters remain high and essentially constant up to the C-9 position of the chain. Beyond the C-11 position, the degreeof order declines rapidly, more so for the axial rotationdetermined by hcos 2�� � ��i than for the off-axis fluctuationthat is determined by hP2�cos �A�i. At the C-14 position inthe terminal methyl region of the chain, the azimuthal orderparameter hcos 2�� � ��i is reduced to zero. Here the �-orientation is completely random, whereas the segmentalorder along the chain-axis direction hP2�cos �A�i ¾ 0.5 is stillappreciable.

Figure 2 also gives the effective maximum angularamplitudes, ˇ and �o, derived from the order parametersby using Eqns (6) and (8). In an inverse fashion, they clearlyreflect the dependences on chain labelling position, n, of thecorresponding order parameters. For the upper part of thechain, up to C-10, the maximum angular excursions of �A

and � are very limited, 30° or less. At the terminal methylregion (C-14), full axial rotation of the chain segments,i.e. �o D 90°, is achieved, but the segmental fluctuation isrestricted to a maximum amplitude ˇ ¾ 50°. The implicationsof these results on chain ordering for the formation ofspatially differentiated membrane domains that are enrichedin cholesterol, as indicated schematically in Fig. 1(b), havebeen discussed previously.3,4

Transmembrane polarity profile and waterpenetrationMeasurements of the gxx-tensor elements are superior – insensitivity to hydrogen bonding and water penetration – to

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s 2(

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)>, o

r <

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<cos 2(f−f)>

<P2(cos qA)>

Tw

ist, fo or tilt, b (°)

fo

Hea

dgro

up

Term

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b

Figure 2. Dependence of the order parameters (solid symbols)and rotational amplitudes (open symbols) on spin-labelposition n in the sn-2 chain of n-PCSL spin probes inmembranes of dimyristoyl phosphatidylcholine + 40 mol%cholesterol at 30 °C. Left-hand ordinate: lateral and transverseorder parameters hcos 2�� � ��i and hP2�cos �A�i, respectively.Right-hand ordinate: azimuthal and off-axis angular amplitudes�o and ˇ, respectively. (Data from Ref. 4).

Copyright 2005 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2005; 43: S20–S25

S22 D. Marsh, D. Kurad and V. Livshits

those of the Azz-hyperfine splitting.2,8 – 10 High-field spec-troscopy therefore offers distinct advantages over conven-tional spin-label EPR for the study of membrane permeabilitybarriers.

Figure 3 shows the polarity profile for one leaflet of abilayer membrane that is composed of DMPC with 40 mol%cholesterol.5 The solid line and symbols are the gxx-tensorelements of the n-PCSL phosphatidylcholines spin-labelledat different positions in the chain. Higher values of gxx

correspond to the lower polarity at the centre of themembrane.

Qualitatively, the shape of the transmembrane profile isthe inverse of that established earlier for the 14N-hyperfineinteraction.12 The dependence of the polarity profiles onspin-label position, n, is of the sigmoidal Boltzmann formintroduced previously:12

gxx�n� D gxx,1 � gxx,2

1 C e�n�no�/� C gxx,2 �9�

where gxx,1 and gxx,2 are the limiting values of gxx at thepolar headgroup and terminal methyl ends of the chainrespectively, no is the value of n at the point of maximumgradient, corresponding to gxx�no� D 1

2 �gxx,1 C gxx,2� and �is an exponential decay constant. Equation (9) represents atwo-phase distribution between membrane regions n > no

and n < no, where the free energy of transfer, �n � no�kBT/�,increases linearly with distance from the n D no plane.

The dotted line and open symbols in Fig. 3 represent theintensities of the 2H-electron spin-echo envelope modulation(ESEEM) from D2O, for the n-PCSL spin labels in membranesof dipalmitoyl phosphatidylcholine C 50 mol% cholesterol.11

The dependence of the D2O profile on position n in themembrane is also of the sigmoidal form described by Eqn (9).Comparison of the high-field results with the 9-GHz ESEEM

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2H am

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(gxx

−ge)

× 1

03

Hea

dgro

up

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3

gxx

D2O amplitude

6

Figure 3. Dependence of the gxx-tensor element (solid line;left-hand ordinate) on spin-label position n in the sn-2 chain ofn-PCSL spin probes in membranes of DMPC C 40 mol%cholesterol at �100 °C. (Data from Ref. 5.) The solid line is anon-linear least-squares fit of Eqn (9) to the g-value data.Dotted line and right-hand ordinate: n-dependence of theelectron spin-echo envelope modulation (2H-ESEEM) fromD2O for n-PCSL in membranes of dipalmitoylphosphatidylcholine C 50 mol% cholesterol. (Data fromRef. 11).

intensities indicates that at least an appreciable – if not thegreater – part of the gxx-profile in Fig. 3 originates from waterpenetration into the membrane.

It is instructive to compare the polarity-induced gxx-shifts with the predictions of quantum chemical calculations.Density functional theory (DFT) calculations by Oweniuset al.13 predict a shift of gxx ³ �4.4 ð 10�4 for one H-bonded water, and gxx ³ �8.2 ð 10�4 for two H-bondedwaters. Semiempirical molecular orbital calculations (PM3,INDO, ZINDO/S) by Plato et al.10 also predict a shift of��4 š 1� ð 10�4 for one H-bond, whereas earlier restrictedopen-shell Hartree–Fock (ROHF) calculations by Engstromet al.14 predicted a shift of only half this value. Taking themore recent theoretical values, the experimental g-shift ofgxx �Dgxx,1 � gxx,2� ³ �6.2 ð 10�4 between the outer (C-4–C-7) and inner (C-10–C-14) regions of the membrane(Fig. 3 and Eqn (9)) corresponds to a reduction in number ofH-bonded water molecules by less than 1.5 per spin label.This upper limit assigns the entire experimental g-shift to H-bonding, ignoring other polarity contributions to the changein gxx. Water (D2O) molecules that are H-bonded to thespin-label nitroxides are observed directly in the 2H-ESEEMmeasurements of Erilov et al.11 From the latter, it is estimatedthat approximately 50% of the nitroxide spin labels in theouter C-4–C-7 regions of the chains are H-bonded to D2O.None are H-bonded in the inner C-10–C-14 regions. The low-field 2H-ESEEM measurements and the high-field g-tensormeasurements are thus in semi-quantitative agreement.

The discrete nature of the H-bonding gives rise toheterogeneity in the amount of water associated with spinlabels at a given position, n < 8, in the upper part of the chain.This manifests itself as inhomogeneous broadening of the gxx-peak in the HF-EPR spectrum. Multi-frequency spectra inFig. 4 illustrate, in principle, the polarity-associated g-strainat the n D 5 position in hydrated membranes. At 360 GHz(for details of the spectrometer, see Ref. 15), the half-width ofthe gxx-feature from 5-PCSL is considerably larger than that at94 GHz. Relative to 9-PCSL, the inhomogeneous broadeningof 5-PCSL increases approximately fourfold between 94 and360 GHz (from 0.37 to 1.5 mT), i.e. scales directly with themicrowave frequency.

The dependence of the polarity-induced inhomogeneousbroadening on position, n, of the spin label in the membraneis illustrated by the 94-GHz spectra of the n-PCSL labelsthat are presented in Fig. 4b.5 The widths of the low-fieldgxx-peak differ greatly between the two regions n < 8 andn > 8. Towards the centre of the membrane, the low-fieldpeaks are remarkably narrow, whereas for positions closeto the top of the chain they are much broader as a result ofthe statistical distribution of water molecules in this regionof the membrane. For the transition region, n D 8, a yetmore dramatic heterogeneity is obvious in that the spectrumconsists of a superposition of components corresponding tothe two flanking regions n < 8 and n > 8.

For 9-PCSL, the lines in the 94-GHz spectrum of Fig. 4are relatively narrow, and the Axx

14N-hyperfine splittingis partially resolved as a very pronounced shoulder onthe high-field side of the gxx-peak. The half-width ofan individual hyperfine line is ca 0.5 mT as measured

Copyright 2005 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2005; 43: S20–S25

High-field spin-label EPR of lipid membranes S23

360 GHzDMPC + 5-PCSL

2.0 mT

12.79 12.80 12.81 12.82 12.83 12.84 12.85 12.86

Field (T)

3.345 3.350 3.355 3.360 3.365 3.370

Field (T)

9-PCSL

8-PCSL

0.85 mT

0.48 mT

5-PCSL

94 GHz

DMPC/Chol (40 mol%)

(a)

(b)

Figure 4. (a) 360-GHz EPR spectrum of 5-PCSL spin-labelledphosphatidylcholine in membranes of DMPC at �100 °C (D.Kurad, M. Fuchs and K. Mobius, unpublished). The small,sharp lines to high field are from the Mn2C/MgO field marker.(b) 94-GHz EPR spectra of n-PCSL spin-labelledphosphatidylcholines in membranes of dimyristoylphosphatidylcholine C 40 mol% cholesterol at �100 °C. (Datafrom Ref. 5.) All spectra are displayed as the first derivative,using field modulation.

on the low-field manifold. For 5-PCSL, the additionalinhomogeneous broadening of the gxx-feature exceeds theAxx hyperfine splitting, and a more symmetrical lineshapewith a half-width of ca 0.85 mT is obtained in the 94-GHzspectrum. The increase of linewidth of 5-PCSL, relative to9-PCSL, corresponds to a distribution width in gxx-strainof υgxx ³ 2 ð 10�4. In terms of the DFT calculations thatwere mentioned earlier,13 this value of υgxx translates, onaverage, to half a water molecule. Considering that a givennitroxide can have up to two strong H-bonds with water,this estimate of the distribution width is not unreasonable.

Multi-frequency EPR to separate fast and slowlipid motionsIncreasing microwave frequency increases the spectralanisotropy and hence decreases the sensitivity of spin-label EPR spectra to slow motions. Spectral simulationsusing the stochastic Liouville formalism demonstrate thatat 94 GHz the principal Azz element of the hyperfine tensoris insensitive to slow off-axial motions with correlation times

½5 ns.16 This is because the Zeeman anisotropy dominatesover the hyperfine anisotropy and, therefore, slow-motionalmodulation shifts all three hyperfine components of the high-field gzz-manifold towards low field, by approximately equalamounts.

A useful parameter for analysing the rapid motions is thehyperfine z-anisotropy ratio:

�A D [hAzzi � 12 �Axx C Ayy�

]/A �10�

This is related to the order parameter hP2�cos �Aloc�i that

characterises the rapid local off-axis motions by:

hP2�cos �Aloc�i D 1

2 �3�A � 1� �11�

which can be derived from Eqn (5), together with theinvariance of the trace of the A-tensor. Whereas the �A

parameter should reflect only fast motions, this is notnecessarily the case for the corresponding parameter:

�g D [hgzzi � 12 �gxx C gyy�

]/g �12�

which is derived from the g-value anisotropy. Only in theabsence of appreciable slow-motional contributions to the94-GHz spectra, is it expected that the �g and �A parametersare equal (cf Eqns (3) and(5)).

Figure 5 shows the dependence of the 94-GHz z-anisotropy ratio, �g/�A, on position, n, of chain labellingfor n-PCSL spin labels in membranes of DMPC C 40 mol%cholesterol, in the liquid-ordered phase at 30 °C.16 Onesees that �g/�A ³ 1 for n D 4–8, but for larger valuesof n the ratio decreases systematically. This means thatfor 4-PCSL to 8-PCSL there is virtually no significantcontribution to the shift of hgzzi from slow overall off-axial tumbling, i.e. that hP2�cos �A

over�i ³ 1 (or the overallmotion is so slow as not to affect hgzzi). This is undoubtedlyattributable to the pronounced effect of cholesterol on chainordering. In contrast, the slow overall off-axis diffusionbecomes appreciable for chain segment positions n > 9.This represents a reduced ordering effect of the rigid sterol

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<P2(cos qA)>

(gxx + gyy)

(Axx + Ayy)∆g∆A

Azz

gzz

rA

rg .−

−=

2121

Figure 5. Dependence of the 94-GHz z-anisotropy ratio �g/�A

(Eqns (10) and (12)) and segmental order parameterhP2�cos �A

loc�i (Eqn (11)) on spin-label chain position n of then-PCSL in membranes of dimyristoyl phosphatidylcholine +40 mol% cholesterol at 30 °C. (Data from Ref. 16).

Copyright 2005 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2005; 43: S20–S25

S24 D. Marsh, D. Kurad and V. Livshits

nucleus, which itself extends approximately down to n ³ 11of the lipid chains.3 Note that the long-axis ordering by theenvironment varies towards the chain end. This representsan increase in detail in describing the chain motion that ispossible from high-field measurements, as compared withthe model of a uniform chain-axis ordering used previouslyto describe n-PCSL spectra obtained at conventional EPRfrequencies (cf Table 1).

Slow-motion effects are manifested much more stronglyin the EPR spectra recorded at low operating frequencies.Combination of the high-field spectra with spectra recordedat 9 GHz, therefore, greatly improves precision in charac-terising the slow off-axial component. From the values ofhP2�cos �A

loc�i that are obtained from �A measured at highfield, one can estimate the value of the g-tensor anisotropyhgiloc that is averaged over the rapid off-axial motion. Thisis done by using Eqn (3). The resulting partially averagedA- and g-values are used to construct a spin HamiltonianhH�over�i that is pre-averaged over the fast local motion.This is then used in the stochastic Liouville equation for thespin density matrix, r:6

∂��over, t�∂t

D �i[hH�over�i, ��over, t�]

� diff�over���over, t� �13�

to describe the slow overall rotation. The latter is specifiedby the diffusion operator diff�over�, which depends on theEuler angles, over, that define the orientation of the axesof rapid local motional averaging, relative to the constantmagnetic field. Simulation of the 9-GHz spectra by usingthis method then yields values of the rotational diffusioncoefficient, DR?over, and order parameter, Szz

over, for theslow-motional component.

Figure 6 shows the dependence on position, n, of the spinlabel in the chain of the order parameter, Szz

over, and diffusioncoefficients, DR?over (and DR//

over), for n-PCSL in membranes

Table 1. Rotational correlation times (�R//, �R? and �J�

obtained by simulations of 9-GHz EPR spectra fromspin-labelled lipid chains, n-PCSL, in oriented membranes ofdimyristoyl phosphatidylcholine17

Position, n T (°C) �R// (s)a �R? (s)a �J (s)b

C-6 25 6.0 ð 10�9 6.0 ð 10�8 1.6 ð 10�11

35 2.7 ð 10�9 2.7 ð 10�8 1.4 ð 10�11

45 1.6 ð 10�9 1.6 ð 10�8 1.3 ð 10�11

C-10 25 6.0 ð 10�9 6.0 ð 10�8 7.2 ð 10�12

35 2.7 ð 10�9 2.7 ð 10�8 6.0 ð 10�12

45 1.6 ð 10�9 1.6 ð 10�8 6.0 ð 10�12

C-13 25 6.0 ð 10�9 6.0 ð 10�8 2.0 ð 10�12

35 2.7 ð 10�9 2.7 ð 10�8 1.7 ð 10�12

45 1.6 ð 10�9 1.6 ð 10�8 1.5 ð 10�12

a Overall long-axis motion, independent of spin-label position.Rotational diffusion coefficients are given by: DR//

over D1/(6�R//) and DR?over D 1/(6�R?).b Local trans–gauche jump rate ��1

J from 2H NMR spectroscopy;spin-label EPR at 9 GHz gives only an upper limit: �J <

2 ð 10�10 s.

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zzloc

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Figure 6. Dependence of the long-axis order parameter Soverzz

(upper panel) and diffusion coefficients DR// and DR? (lowerpanel) for the slow overall motion on spin-label chain position nof the n-PCSL in membranes of dimyristoylphosphatidylcholine C 40 mol% cholesterol at 30 °C. Valuesare deduced from combined measurements at 9 and 94 GHz.(Data from Ref. 16).

of DMPC C 40 mol% cholesterol, in the liquid-ordered phaseat 30 °C.16 The low values of the diffusion coefficients confirmthat the overall motion of the lipid chains is in the slow regimeat 9 GHz and would have relatively minor influence on thespectra at 94 GHz. This justifies the method used for thetime separation in Eqn (13). From Fig. 6 it is seen that theorder parameter, Szz

over, corresponding to the slow overallmotional mode gradually decreases with increasing n downthe chain. The figure also shows that as the amplitude ofthe slow overall motion increases (i.e. Szz

over decreases), sodoes the corresponding rotational rate given by DR?over (andalso DR//

over). As mentioned already, the overall motion ofthe chain axis cannot be represented simply as that of arigid rod. This is not entirely surprising for a flexible chain,and represents a varying effect of the chain environment onproceeding deeper into the membrane.

EXPERIMENTAL

Experimental details are presented fully in previouspublications.4,5 Essential points only are summarised here.EPR spectra were recorded at a microwave frequency of94 GHz on a Bruker E680 heterodyne W-band EPR spec-trometer with a TE011-mode cylindrical cavity resonator anda split-coil superconducting magnet. Low-field spectra wereobtained on a Bruker EMX 9-GHz spectrometer operatingwith a rectangular cavity in the conventional continuous-wave mode. Fully hydrated (100 mM KCl, 10 mM Tris, 1 mM

Copyright 2005 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2005; 43: S20–S25

High-field spin-label EPR of lipid membranes S25

EDTA, pH 7.5), spin-labelled (0.5 mol%) lipid dispersionswere introduced into 0.5 or 0.2 mm i.d. quartz capillar-ies for 94-GHz measurements, or into 1-mm diameter glasscapillaries for 9-GHz measurements. Samples were then con-centrated by centrifugation, the excess aqueous supernatantwas removed, and the capillaries were flame-sealed.

Experiments at 360 GHz were performed on a heterodyneinduction-mode spectrometer with quasi-optics that wasconstructed in the Institute for Experimental Physics atthe Free University of Berlin. This spectrometer has beendescribed in detail by Fuchs et al.15 The spin-labelled lipidsample was deposited as a thin film on the mirror of theFabry-Perot resonator.

Bilayer membranes were formed from the phospho-lipid DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine)obtained from Avanti Polar Lipids (Alabaster, AL). Phos-phatidylcholines spin-labelled at C-atom n of the sn-2 chain(1-acyl-2-[n-(4,4-dimethyloxazolidine-N-oxyl)]stearoyl-sn-glycero-3-phosphocholine; n-PCSL) were synthesised asdescribed by Marsh and Watts.18

Spectral EPR simulations for nitroxide spin labels under-going slow Brownian rotational diffusion in an orientingpotential were performed according to the stochastic Liou-ville formalism.19 Simulation was implemented by using thesoftware described by Schneider and Freed20 with extensionsby Budil et al.21

AcknowledgementsAll measurements at 94 GHz in the papers reviewed here wereperformed in collaboration with Dr. Gunnar Jeschke in his laboratoryat the Department of Prof. Hans-W. Spiess, Max-Planck-Institut furPolymerforschung, Mainz. Preliminary spectra at 360 GHz that areillustrated in Fig. 4(a) were recorded by Dr. Martin Fuchs, on thespectrometer built in the laboratory of Prof. Klaus Mobius at theInstitut fur Experimentalphysik der Freien Universitat Berlin. Weare deeply grateful for all these generous collaborations. All thehigh-field EPR work reviewed here was performed as part of thePriority Programme ‘High-Field EPR in Biology, Chemistry and

Physics’, financed by the Deutsche Forschungsgemeinschaft, whomwe also thank.

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2. Marsh D, Kurad D, Livshits VA. Chem. Phys. Lipids 2002; 116: 93.3. Kurad D, Jeschke G, Marsh D. Appl. Magn. Reson. 2001; 21: 469.4. Kurad D, Jeschke G, Marsh D. Biophys. J. 2004; 86: 264.5. Kurad D, Jeschke G, Marsh D. Biophys. J. 2003; 85: 1025.6. Lou Y, Ge M, Freed JH. J. Phys. Chem. B 2001; 105: 11 053.7. Livshits VA, Marsh D. In Very High Frequency (VHF) ESR/EPR,

Grinberg OY, Berliner LJ (eds). Kluwer Academic: New York,2004; 431.

8. Kawamura T, Matsunami S, Yonezawa T. Bull. Chem. Soc. Jpn.1967; 40: 1111.

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