Proc. Nati. Acad. Sci. USAVol. 84, pp. 3704-3708, June 1987Biophysics

Integral membrane proteins significantly decrease the molecularmotion in lipid bilayers: A deuteron NMR relaxation study ofmembranes containing myelin proteolipid apoprotein

(lipid-protein interaction/multipulse dynamic NMR/order parameters/rotational correlation times)

P. MEIER*, J.-H. SACHSEt, P. J. BROPHYt, D. MARSHt, AND G. KOTHE**Institut fur Physikalische Chemie der Universitat Stuttgart, Pfaffenwaldring 55, D-7000 Stuttgart 80, Federal Republic of Germany; tMax-Planck-Institut furbiophysikalische Chemie, Am Fassberg, D-3400 Gottingen-Nikolausberg, Federal Republic of Germany; and *Department of Biological Science, StirlingUniversity, Stirling FK9 4LA, United Kingdom

Communicated by Harden M. McConnell, February 9, 1987

ABSTRACT The influence of the myelin proteolipidapoprotein on lipid chain order and dynamics was studied by2H NMR of membranes reconstituted with specifically deuter-ated dimyristoyl phosphatidylcholines. Quadrupolar echo andsaturation recovery experiments were fitted by numericalsolution of the stochastic Liouville equation, using a model thatincludes both inter- and intramolecular motions [Meier, P.,Ohmes, E. & Kothe, G. (1986) J. Chem. Phys. 85, 3598-3614].Combined simulations of both the relaxation times and thequadrupolar echo line shapes as a function of pulse spacingallowed unambiguous assignment of the various motionalmodes and a consistent interpretation of data from lipidslabeled on the C-6, C-13, and C-14 positions of the sn-2 chain.In the fluid phase, the protein has little influence on either thechain order or the population of gauche rotational isomers butstrongly retards the chain dynamics. For 1-myristoyl-2-[13-2H2]myristoyl-sn-glycero-3-phosphocholine at 35°C, the corre-lation time for chain fluctuation increases from 20 nsec to 650nsec and for chain rotation from 10 nsec to 180 nsec, and thegauche isomer lifetime increases from 0.15 nsec to 1.75 nsec, ongoing from the lipid alone to a recombinant of protein/lipidratio 0.073 mol/mol. The results are essentially consistent withspin-label ESR studies on the same system [Brophy, P. J.,Horvath, L. I. & Marsh, D. (1984) Biochemistry 23, 860-865],when allowance is made for the different time scales of the twospectroscopies.

Lipid-protein interactions are important determinants ofbiological membrane structure and function and, for thisreason, have been the subject of intensive study by physi-cochemical methods. Magnetic resonance spectroscopy hasmade major contributions in this area because of its uniquesensitivity to anisotropic molecular motion. In consequenceof the different time scales of the two spectroscopies, nuclearmagnetic resonance (NMR) and electron spin resonance(ESR) are sensitive to different aspects of the lipid dynamics,and this has given rise to rather divergent views of thelipid-protein interaction. The ESR spectra of spin-labeledlipids interacting with integral membrane proteins have beeninterpreted in terms of two components in slow exchange, onthe nanosecond time scale. One component is characteristicof the lipid mobility in fluid, protein-free bilayers, and theother corresponds to lipids whose mobility is restricted bydirect interaction with the intramembranous hydrophobicsurface of the protein. The two-component nature of thespectra has allowed direct study of the stoichiometry andspecificity of the interaction in a wide variety of different

lipid-protein systems (see ref. 1 for review). The 2H NMRspectra of deuterated lipids, by contrast, have been found toconsist of a single component in various lipid-protein sys-tems, indicating that all lipid populations are in fast exchange,on the millisecond time scale. 2H NMR investigations havetherefore concentrated on the ordering of the lipid chains. Itis found that there is little or no change in chain ordering,although spectral line broadening does indicate a reduction inthe rate of chain motion (e.g., see refs. 2 and 3).The complexity of the systems involved dictates that a

detailed description of the effects of integral membraneproteins on lipid chain dynamics can best be achieved by acombination of multipulse NMR experiments with compre-hensive theoretical simulations. Such an analysis is currentlylacking. In the present work, we have investigated myelinproteolipid apoprotein reconstituted with specifically deuter-ated dimyristoyl phosphatidylcholine ([Myr2]PtdCho) as amodel system for lipid-protein interactions in biologicalmembranes. A motional model has been employed thatincludes both inter- and intramolecular motion (i.e., bothlong-axis motion and trans-gauche isomerization) and whichis valid in both fast and slow motional regimes (4, 5). Thesimulation of quadrupole echo spectra as a function of pulsespacing, together with measurements of spin-lattice relax-ation time (Tz), allows discrimination of the different mo-tional modes, leading to an unambiguous description of themolecular dynamics. A consistent interpretation is obtainedof data from reconstitutions with [Myr2]PtdCho labeled at theC-6, -13, and -14 atoms of the sn-2 chain. It is found that theprotein has very little effect on either the degree of order ofthe lipid chain or the population of gauche rotational isomersbut increases the rotational correlation times for chainfluctuation, chain rotation, and trans-gauche isomerism by afactor of 10 or more. These results are fully consistent withthose obtained from ESR spectroscopy of spin-labeled lipids(6, 7).

THEORYAnalysis of the various 2H NMR relaxation experiments wasperformed using a comprehensive motional model, describedin refs. 4 and 5. The basis of the model is the density matrixformalism. The action of the different radiofrequency (if)pulses on the density matrix p(t) is represented by unitarytransformations, involving Wigner rotation matrices. Be-

Abbreviations: PtdCho, phosphatidylcholine; [Myr2]PtdCho,dimyristoyl phosphatidylcholine; [Myr2]PtdCho-n-d2, 1-myristoyl-2-[n-2H2]myristoyl-sn-glycero-3-phosphocholine; [Myr2]PtdCho-14-d3, 1-myristoyl-2-[14-2H3]myristoyl-sn-glycero-3-phosphocholine.

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tween pulses the density matrix is assumed to obey thestochastic Liouville equation (8, 25)

-P(flt) =at

-(i/h ) HX(fl).p(,t) - r'flp(Qft) - Peq(fl)]b [1]

which is solved using a finite grid method (9, 26). HX(f) is theHamiltonian superoperator of the spin system, which de-pends on the orientation and conformation of the molecule,specified by the Euler angles fQ. 1F is the time-independentMarkov operator for the various motional processes, wherethe equilibrium distribution Peq(Q) is given by

FflPeq (4) = 0 [2]

and Peq(Q) is the equilibrium density matrix.The Markov operator includes both intermolecular and

intramolecular motions. The intermolecular motion is theanisotropic rotational diffusion of the lipid molecule as awhole, within an orienting potential. The intramolecularchain motion consists of trans-gauche isomerization, whichis represented by ajump process. The dynamics ofthe systemare characterized by three correlation times: TRI, and TRR forrotation about the diffusion tensor axis and rotation of thisaxis, respectively, and Tj for trans-gauche isomerization.The equilibrium distribution Peq(Q) is described in terms ofinternal and external coordinates. The internal part accountsfor different conformations, and the external part for differentorientations. Generally, there are four different conforma-tional states corresponding to the different allowed orienta-tions of a particular chain segment relative to the chain axis.The Euler angles OK,K (K = 1, 2, 3, 4) characterizing theseorientations are listed elsewhere (4, 5). The conformationalpopulations may be used to set up a segmental order matrix(4), which on diagonalization yields the segmental orderparameters Sz z and Sxx - Sy r . They express the orderingof the most-ordered segmental axis Z' and the anisotropy ofthe order, respectively.The orientational distribution of the phospholipid mole-

cules is described in the mean-field approximation, using anorienting potential from the molecular theories of liquidcrystals (10). For axially symmetric ordering the normalizeddistribution function

f(f) = Njexp(A cos23) [3]

depends on a single parameter A, characterizing the orien-tation of the molecules with respect to the local director. Theorientational order parameter, Szz, is related to the coeffi-cient A by a mean value integral:

r

Szz = - N1 j [3 (cos2p8) - 1]exp(A cos2/3)sin P d, [4]2 0

The molecular order of the phospholipid molecules is thusspecified by the orientational order parameter Szz and thesegmental order parameters Sz'z and Sxyx - Syry.The time evolution ofthe density matrix for a general pulse

sequence is set up using the appropriate Wigner rotationmatrices and the equation of motion. Before an rf pulse isapplied, the spin system is at thermal equilibrium, Peq, witha Boltzmann population of the spin states. Application of thefirst pulse creates a defined nonequilibrium state p(O). Afterthe pulse, the density matrix evolves according to thestochastic Liouville equation (Eq. 1). A second pulse is thenapplied, preparing a new initial condition, followed by a

second evolution period and so on. Finally, after the nthpulse, the observable NMR signal is given by

L(t, T1, T2, . . Tn-1) = Tr[p(t, r1, r2, . - * Tn-l)-I+], [5]

where I+ is the nuclear spin-raising operator and ri, r2 . . .

Tnri are the various pulse-separation times. Fourier trans-formation of L(t, rT, T2, . . Tn-1) starting from time t = r1 +72 . . * + Tn-i + Ti corresponding to a spin echo, yields singlequantum spectra, which depend sensitively on the actualpulse sequence. From the decay of the echo amplitude as afunction of i, various relaxation times can be evaluated.Two different pulse sequences are mainly employed in this

study: the quadrupole echo sequence, (X/2)x - T - (f/2)y,and the saturation recovery sequence, (X/!2) - T1 - (X/2)x -T2- (iT/2)y (4, 5). Since the echo spin-spin relaxation time,T2E, is dominated by motions with correlation times of theorder of the inverse of the 2H quadrupole coupling constant,TR- (e2qQ/h )-1, the quadrupole echo sequence is sensitiveto motions in the range 10-8 sec < TR < 10-4 sec. Saturationrecovery is determined by the spin-lattice relaxation times,Tlz, which are particularly sensitive to motions with corre-lation times at the inverse ofthe Larmor frequency, TR 1.For a field strength ofB = 7.0 T, this corresponds to overallsensitivity in the range 10-1" sec < TR < 10-7 sec. Thus, bycombining quadrupole echo and saturation recovery studies,it is possible to follow dynamic processes over 7 orders ofmagnitude of correlation times.

MATERIALS AND METHODSMaterials. Myelin was isolated from bovine spinal cord

(11). The proteolipid was extracted and delipidated bychromatography on Sephadex LH-20 in CHC13/CH30H/0.1M HC (50:50:1, vol/vol) as described (12). Chromatographywas repeated in order to ensure complete delipidation, asjudged by thin-layer chromatography and phosphate analy-sis.

[6,6-2H2]- and [13,13-2lH2myristic acid were synthesizedessentially as described (13). Acetic acid hexyl ester (ornonanoic acid ethyl ester) was reductively deuterated withLiAl2H4, and the deuterated alcohol was then converted tothe bromide, as starting material for a Grignard reaction. Theappropriate w-bromo fatty acid was converted to its chloro-magnesium salt by using CH3MgCl. This was then coupled tothe deuterated Grignard reagent, using a LiCuCl4 catalyst intetrahydrofuran at -130C. [13,13-2H2]myristic acid was usedto acylate sn-1-myristoyl lysophosphatidylcholine accordingto ref. 14, to yield [Myr2]PtdCho deuterated on the C-13 atomof the sn-2 chain ([Myr2]PtdCho-13-d2). The lysophospha-tidylcholine was prepared from [Myr2]PtdCho by phospho-lipase A2 digestion, under conditions for the activity assaygiven by the suppliers (Boehringer Mannheim). [Myr2]Ptd-Cho deuterated on the C-6 and C-14 atoms of the sn-2 chain([Myr2]PtdCho-6-d2 and [Myr2]PtdCho-14-d3) were synthe-sized as described in ref. 4.Sample Preparation. The proteolipid protein was reconsti-

tuted with the specifically deuterated [Myr2]PtdCho by dial-ysis from 2-chloroethanol as described (12). The buffer usedthroughout was 0.1 M NaCl/1 mM EDTA/2 mM Hepes, pH7.5. Samples in 2-chloroethanol (volume, 100-300 ml) weredialyzed against five changes of 5 or 10 liters (minimum30-fold excess) of buffer at 4°C. Dialyzed samples wereanalyzed by sucrose density gradient centrifugation [10-55%(wt/vol) sucrose in buffer; Beckman SW40 rotor, 40,000 rpm,3 hr] and recovered essentially as a single band. Lipidphosphorus (15) and protein content (16) of the reconstitutedcomplexes were determined as described.For NMR measurements, the reconstituted complexes

were pelleted (61,700 x g, 45 min), resuspended and washed

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four to five times in buffer prepared with deuterium-depletedwater, and finally packed into 1-ml (8-mm-diameter) poly-ethylene sample tubes.NMR Methods. 2H NMR experiments were performed at

46.1 MHz with a Bruker CXP 300 spectrometer, usingquadrupole echo and saturation recovery sequences. The ff/2pulse width was 4 tusec, with a home-built probe (10-mmcoil). All experiments were recorded using quadrature de-tection with a digitizing rate of up to 2 MHz and appropriatephase-cycling schemes (4, 5). The number of scans variedbetween 3600 and 36,000 (3-Hz repetition rate), with samplescontaining about 75 mg of deuterated lipid.

Simulations of multipulse dynamic NMR experiments forI = 1 spin systems undergoing inter- and intramolecularmotion in an anisotropic medium were performed as de-scribed (4, 5). The spin Hamiltonian for the Zeeman andquadrupole interactions includes nonsecular contributions.Within the Redfield limit (17), analytical expressions wereused in the analysis. The constant parameters in the calcu-lations were obtained from fast-rotational and rigid-limitquadrupole echo spectra of the pure phospholipids. As foundpreviously (4), the quadrupole coupling constant of thealiphatic deuterons is e2qQ/h = 169 kHz. Residual quadru-polar echo relaxation times of TOE = 1 msec ([Myr2]PtdCho-6-d2) and TOE = 2.5 msec ([Myr2]PtdCho-13-d2, [Myr2]Ptd-Cho-14-d3) were used to account for the effect of dipolarinteractions, which are omitted from the spin Hamiltonian.The adjustable parameters Szz, Szz,, (Sx'x' - Srr), TRii,

TRI, and Ti (see Theory) were determined by simulation of thevarious experiments. In general, they need not be variedindependently. In the fast-motional region, Szz, Sz'z,, and(Sxx, - Syry) can be obtained from the splittings of thequadrupole echo spectra (4). The dynamic parameters TRII,TR±, and Tj are then reliably evaluated by analyzing thedifferent pulse experiments.

RESULTSRepresentative quadrupolar echo 2H NMR spectra of [Myr2]-PtdCho-13-d2 in recombinants of different protein/lipid ra-tios, in the fluid phase, are given in Fig. 1. Clearly, both thespectral line shapes and the angular dependence of thequadrupolar echo relaxation time, T2E (as indicated by thechange in line shape as a function of the pulse spacing, rl, inthe quadrupolar echo sequence), show a very marked de-pendence on the protein/lipid ratio. Quadrupolar echo spec-tra of recombinants with the same protein/lipid ratio, butwith different positions of the deuterium label on the sn-2chain of the [Myr2]PtdCho, in the fluid phase, are given inFig. 2. These spectra show clear sensitivity to the differentialsegmental motion at the various chain positions, in thepresehce of the protein. In all cases the spectra consist of asingle component, with no evidence for a second, broader,underlying component.The dependence of the quadrupolar echo relaxation time,

T2E (defined by the pulse spacing, ri, at which the totalintegrated intensity of the quadrupolar echo spectrum isreduced to l/e of its initial value), and the spin-latticerelaxation time, Tlz, on the protein/lipid ratio for recombi-nants with [Myr2]PtdCho-13-d2 is given in Fig. 3. Therelaxation curves were single-exponential decays, corre-sponding to just one spectral component, in all cases. Boththe quadrupolar echo and spin-lattice relaxation times de-crease strongly with increasing protein content in the recom-binant, indicating a very pronounced effect of the protein onthe lipid chain motion.Combination of the dependence of the echo line shapes on

pulse spacing in Fig. 1, together with the measured relaxationtimes in Fig. 3, allows separation of the contributions fromthe different intra- and intermolecular chain motions, as

10 kHza b c

I'

b J

FIG. 1. Experimental (-) and simulated (---) 2H NMR spectra ofmyelin proteolipid apoprotein/[Myr2]PtdCho-13-d2 recombinants atdifferent protein/lipid ratios, at 350C. All spectra refer to quadrupoleecho sequences. (Top) Pure lipid (protein/lipid ratio = 0) with Tr =

60 Asec (spectra a), 270 usec (spectra b), or 600 tusec (spectra c).(Middle) Protein/lipid ratio = 0.033 mol/mol, with T1 = 60 usec(spectra a), 120 ,usec (spectra b), or 270 psec (spectra c). (Bottom)Protein/lipid ratio = 0.07 mol/mol with T = 60 pisec (spectra a), 90Asec (spectra b), or 120 Asec (spectra c). The simulations wereobtained with the parameters of Fig. 4. Spectra were normalized toreflect the actual intensity in a quadrupole echo sequence.

indicated in ref. 4. The simulated spectra (dashed lines),obtained by using a single set of motional parameters for eachprotein/lipid ratio, are compared with the experimentalspectra for the [Myr2]PtdCho-13-d2 lipid in Fig. 1. There is a

generally good agreement between experiment and simula-tion, in terms of both line shapes and intensities, throughoutthe quadrupolar echo sequences. The calculated dependenceof the relaxation times on protein/lipid ratio is given by thesolid lines in Fig. 3. The spin-lattice relaxation times werefitted independently, using Redfield theory, and serve todetermine the values for the fast correlation time, Tj. Onlywhen Tj - 1 nsec, does this parameter influence thequadrupolar echo line shapes, and in this region the twomethods give values that are in good agreement. Thequadrupolar echo relaxation times are determined complete-ly by the parameters from the line shape and intensitysimulations in Fig. 1. The good agreement between experi-mental and calculated relaxation times in Fig. 3 thereforefurther demonstrates the consistency ofthe description oftheline shapes and relaxation behavior. Simulation of thequadrupolar echo spectra for the different label positions isgiven in Fig. 2 (dashed lines). In this case, exactly the samesimulation parameters were used for the three different labelpositions, with the exception of the segmental order param-eters for the [Myr2]PtdCho-6-d2 lipid. (The values ofthe latterare very close to those for the lipid alone at 350C: Sz'z = 0.75,Sxx - Sryy = 0.12). This again confirms the consistency ofthe motional model, since all other motions should beindependent of label position, and the motions at the 14position are conformationally predictable from those ofthe 13position.The protein/lipid ratio dependence of the motional corre-

lation times and segmental order parameters, deduced fromthe integrated description of the spectral parameters for the[Myr2]PtdCho-13-d2 lipid, are given in Fig. 4. It is clear thatalthough the protein has virtually no effect on the chain order,the rates of all the different modes of chain motion are

c

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KI b C

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Proc. Natl. Acad. Sci. USA 84 (1987) 3707

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FIG. 2. Experimental (-) and simulated (---) 2H NMR spectra of[Myr2]PtdCho-6-d2, [Myr2]PtdCho-13-d2, and [Myr2]PtdCho-14-d3 inrecombinants of similar protein/lipid ratios, at 35TC. All spectra referto quadrupole echo sequences. (Top) [Myr2]PtdCho-6-d2 (protein/lipid ratio = 0.039 mol/mol) with T1 = 30 psec (spectra a), 60 ,Asec(spectra b), or 90 pisec (spectra c). (Middle) [Myr2]PtdCho-13-d2(protein/lipid ratio = 0.04 mol/mol) with Tr = 60 ,usec (spectra a), 90Asec (spectra b), or 120 jusec (spectra c). (Bottom) [Myrj]PtdCho-14-d3 (proteih/lipid ratio = 0.039 mol/mol) with T1 = 30 psec (spectraa), 90 Asec (spectra b), or 180 Asec (spectra c). The simulations wereobtained with the parameters of Fig. 4, except for Szz = 0.73 andSxx - Sr = 0.11 in the case of [Myr2]PtdCho-6-d2. Spectra werenormalized to reflect the actual intensity in a quadrupole echosequence.

strongly influenced by the lipid-protein interaction. Theresults from the [Myr2]PtdCho-6-d2 and tMyr2]PtdCho-14-d3lipids are also consistent with this conclusion (Fig. 2), Sincelittle change is found in the various order parameters, but the

2 4 6Protein/lipid ratio (mol/mol) x 10-2

E

12.5

FIG. 3. 2H quadrupolar echo relaxation times T2E (O, left ordi-nate) and spin-lattice relaxation times Tjz (i, right ordinate) of[Myr2]PtdCho-13-d2 as a function of protein/lipid ratio in the recom-binant. Symbols denote experimental values taken from the inte-grated absorption powder pattern. Solid lines represent calculationsof the relaxation times, using the parameters of Fig. 4. Temperature,350C.

0

ES.

0.

Protein/lipid ratio (mol/mol) x 10-2

FIG. 4. (Upper) Rotational correlational times characterizing theinter- and intramolecular dynamics of [Myr2]PtdCho-13-d2 in myelinproteolipid recombinants, as a function of protein/lipid ratio. Circlesrefer to chain fluctuation (TR,), triangles denote chain rotation (TRII),and squares refer to trans-gauche isomerization (Tj). Temperature,350C. (Lower) Order parameters characterizing orientational andconformational order of [Myr2]PtdCho-13-d2 in myelin proteolipidrecombinants, as a function of protein/lipid ratio. Circles denote theorientational order parameter Szz, squares refer to the segmentalorder parameter Szz, and diamonds denote the anisotropy Sx, -

Srr of the segmental order. Temperature, 350C.

rotational correlation times are much increased. [For the purelipid, very little difference is found in the motional parame-ters for the different labels in the fluid phase, with theexception of the segmental order parameters, Szz,, Sr y, andSxyxy (see ref. 4), Therefore, the parameters Szz, TRfl, TRL,and Tj at a protein/lipid ratio = 0 in Fig. 4 can be taken asrepresentative for all three positional isomers.]

DISCUSSIONIn the fluid phase, the 2H NMR spectra of the deuterium-labeled lipids all consist of a single component and displaysingle-component relaxation times, indicating either a homo-geneous lipid environment or rapid exchange via lateraldiffusion (at a rate > 106 sec-1) between environments ofdiffering lipid mobility. ESR experiments on the same sam-ples, using a [Myr2]PtdCho probe spin-labeled on the 13

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position of the sn-2 chain (data not shown), have all yieldedtwo-component spectra, with the proportion of the moreNotionally restricted component increasing with increasingprotein content of the recombinant. These latter results are infull agreement with previous ESR studies on the proteolipidapoprotein/[Myr2]PtdCho system, obtained using PtdChospin-labeled on the 14 position of the sn-2 chain (6). The ESRresults therefore indicate an inhomogeneous lipid population,but with exchange between the component populations thatmust be fast enough to give averaging of the NMR spectra.Indeed, recent ESR estimations of the exchange rate of thelipids on and off the surface of the myelin proteolipidapoprotein have yielded values 107sec' for PtdCho, whichwould ensure fast exchange on the NMR time scale (7).The NMR results have been successfully interpreted in

terms of a single-component motional model in which thelipid chain dynamics are progressively perturbed by increas-ing protein content. An unambiguous discrimination of thevarious motional modes has been achieved by a comprehen-sive analysis of the effects on the spin dynamics in thedifferent pulse sequences, as described previously for purelipid systems (4). Strikingly, a consistent description can begiven for the line shapes of all three different positionalisomers, even though those for [Myr2]PtdCho-13-d2 and[Myr2]PtdCho-14-d3 in no way approximate to a conventionalPake doublet, as has been assumed in most previous (mo-tional-narrowing) analyses (2, 3).Both the segmental and orientational lipid chain order

parameters are essentially unaffected by the lipid-proteininteraction (Fig. 4 Lower), indicating that the ensembleaverage chain orientation and gauche rotational isomer pop-ulation at the protein surface are not appreciably differentfrom those in fluid lipid bilayers. Similar qualitative conclu-sions have been reached from 2H NMR studies on myelinproteolipid apoprotein (18) and a variety of other lipid-protein systems (2, 3), although these previous analyses havenot explicitly distinguished chain order from chain dynamics.ESR experiments have suggested that the spin-labeled lipidchains associated with the protein do not possess a highdegree of orientational order (19, 20), in qualitative agree-ment with the present results. In contrast, fluorescent-probemeasurements on the other systems have been interpreted tosuggest that an increase in order of the lipid chains is inducedby the protein (21, 22), contrary to the present directmeasurements. The boundary conditions imposed on thechain order parameters in the fluid phase by the presentmeasurements will clearly have important implications forthe interpretation of theoretical models of lipid-proteininteractions (23).The protein has a very strong effect on the lipid chain

dynamics; all correlation times are increased by a factor of 10or more at the high protein contents (Fig. 4 Upper). Clearly,both the chain-axis motion and the trans-gauche isomerismare slowed down at the protein interface, but with preserva-tion of the differential rates between the inter- and intramo-lecular motions. The degree of restriction of chain motionmay be greater than that for other integral proteins, at leastfor the faster motions, since the effects on Tjz are greater.Typically, integral proteins have been reported to decreaseTjz by 20-30% (2, 3), whereas at high concentrations theproteolipid protein decreases Tjz by up to a factor of 5 (Fig.3). ESR measurements on the proteolipid apoprotein/[Myr2]PtdCho system are in qualitative agreement with theNMR results, in that the effective rotational correlation timeof the motionally restricted lipids is a factor of =10 longerthan that of the fluid lipids (6), although in the ESR case adetailed analysis of the chain dynamics was not possible.The protein/lipid-ratio dependence of the NMR-derived

correlation times plateaus at a protein/lipid ratio -0.073

mol/mol, which corresponds quite closely to the number ofNotionally restricted lipids per protein found previously byESR (6). This latter value also correlates well with theestimated number of lipids that can be accommodated aroundthe protein hexamer that is found in detergent-solubilizedproteolipid apoprotein (24). The detailed protein/lipid-ratiodependence of the correlation times in Fig. 4 is not expectedto conform to a strict two-component model for fast ex-change, since it will also contain contributions from pertur-bations of the mobility of lipids beyond the first shellsurrounding the protein. Nevertheless, the motional param-eters measured at the higher protein/lipid ratios are expectedto approximate reasonably closely those of the lipids at theprotein interface and provide a rather complete description ofthe way in which the lipid chain mobility is restricted by theprotein. The rates of all chain motions are reduced by a factorof 10 or more. It remains for future work to determine to whatextent these results for myelin proteolipid apoprotein may begeneralized to other integral membrane proteins.

We thank Frau S. Schreiner and Mrs. S. Chattejee for their experttechnical assistance in the sample preparation. This work wassupported by a grant from the Multiple Sclerosis Society of GreatBritain and Northern Ireland to P.J.B. and from the DeutscheForschungsgemeinschaft to G.K.

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8. Kubo, R. (1969) in Stochastic Processes in Chemical Physics,Advances in Chemical Physics, ed. Shuler, K. E. (Wiley, NewYork), pp. 101-127.

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27, 565-570.12. Brophy, P. J. (1977) FEBS Lett. 84, 92-95.13. Das Gupta, S. K., Rice, D. M. & Griffin, R. G. (1982) J. Lipid Res.

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