quantitative determination of conformational disorder in the acyl chains of phospholipid bilayers by...

7/28/2019 Quantitative Determination of Conformational Disorder in the Acyl Chains of Phospholipid Bilayers by Infrared Spe

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8934 Biochemistry 1989, 28, 8934-8939Kirk, G. L., Gruner , S . M. , & Stein, D. L. (1984) Biochem-istry 23, 1093-1102.Knoll, W., Schmidt, G., Ibel, K., & Sackmann, E. (1985)Biochemistry 24, 5240-5246.Laggner, P. (1984) in Structural Biological Applications ofX-ray Absorp tion, Scattering and Diffraction (Chance, B.,& Bartunik, H. D. , Eds.) pp 171-182, Academic Press,London.Murari, R., Murari, M. P., & Baumann, W. J . (1986) Bio-chemistry 25, 1062-1067.

Navarro, J . , Toivio-Kinnucon, M., & Racker, E. (1984)Biochemistry 23, 130-1 3 5 .Reich, M. K., Kam, Z., & Eisenberg, H. (1982) Biochemistry21, 5189-5195.

Seddon, J. M., Cevc, G., & Marsh, D. (1983) BiochemistryShipley, G. G . (1973) in Biological Membranes (Chapman,D. , & Wallach, D. F. M., Eds.) Vol. 2, pp 1-89, AcademicPress, London.Valtersson, C. , van Duyn, G., Verkleij, A. J. , Chojnachi, T. ,De Kruijff, B., & Dallner, G. (1985) J . Biol. Chem. 260,Verkleij, A . J. (1984) Biochim. Biophys. Acta 779, 43-63.Wachtel, E., & Bach, D. (1987) Biochim. Biophys. Ac ta 922,Wieslander, A., Rilfors, L., & Lindblom, G. (1986) Bio-Yeagle, P. L. (1985) Biochim. Biophys. Acta 822,267-287.

22, 1280-1 289.

2742-275 1.

234-23 8.chemistry 25, 751 1-7517.

Quantitative Determination of Conformational Disorder in the Acyl Chains ofPhospholipid Bilayers by Infra red Spectroscopy+R. Mendelsohn,*J M. A. Davies ,$ J . W . Brauner ,l H. F. Schus ter , t and R. A . Dluhy*,g

Department of Chem istry, Rutgers University, 7 3 Warren Stre et, Newark, New Jersey 07102, and National Center o rBiomedical Infrared Spectroscopy, Battelle M emorial Institute, 505 King Avenue, Columbus, Ohio 43201Received May 18 , 1989; Revised Manuscript Received June 30, I989

ABSTRACT: A method is proposed and demonstrated for the direct determination of conformational disorder(trans-gauche isomerization) as a function of acyl-chain position in phospholipid bilayer membranes. Th re especifically deute rate d derivatives of dipalmitoylphosphatidylcholine (D PP C) , namely 4,4,4,4-d4-DPPC(4-d 4-D PP C), 6,6,6,6-d4-DPPC (6-d 4-D PP C), and 10,10,10, 10-d4-DPPC (10-d4-DPPC ), have beensynthesized. Th e CD 2 rocking modes in the Fourier t ransform infrared ( FT -IR ) spectrum have beenmonitored as a function of tempe ratu re for each derivative. A method originally applied by Snyd er andPoore [(1973) Macromolecules 6,708-7151 as a specific probe of hydrocarbon chain conformation in alkaneshas been used to analyze the da ta. Th e rocking modes appear a t 622 cm-* for a C D2 segment surroundedby a tran s C-C-C skeleton and between 645 and 655 cm- for segments surrounded by particular ga uch econfo rmers . Th e integrated band intensities of these modes have been used to monitor trans-gaucheisomeriza tion in the acyl chains at particula r de pth s in the bilayer. At 48 OC, above the gel-liquid-crystalphase t ransi tion, the percentage of gauc he rotam ers present is 20.7 f .2, 32.3 f .3, and 19.7 f .8 for4-d4-D PPC , 6-d4-D PPC , and 10-d4 -DP PC, espectively. Th e gel phase of the latter two molecules is highlyordered. In contrast , a substant ial populat ion of gauche rotamers was observed for the 4-d4-D PPC . T heconformational analysis yields a range of 3.6-4.2 gauche rotam ers/acyl chain of DP PC above the phasetransition. This range is in excellent acco rd with the dilatometric da ta of Na gle and Wilkinson [(1978 )Biophys. J. 23, 159-1751, Th e significant adva ntages of the FT-I R approac h are discussed.

Determination of the structure and dynamics of phospho-lipids, and the relationship of these qu antities to the fu nctionof membrane proteins, is a long-sought goal of membranebiophysics. Toward this end, the arsenal of modern spectro-scopic technology has been brought to bear. Each techniquecontributes information on its own time scale to a compositepicture of phospholipid organiz ation. Yet a qua ntitati vepicture of the co ntributio n of any single motion to phospholipiddynamics is elusive. Th e reason for this is not difficult tounderstand . For example, the most powerful techniquebrought to bea r on this problem to date is probably *H N M R

Supported by N IH Gr ants GM-29864 (R.M.) and RR-01367*Address correspondence to these authors.* Rutgers University.8 Battelle Memorial Institute.

(R.A.D.) .

spectroscopy [for a recent review, see Seelig and M acDonald(1987)l. The order parameters derived from this experimentincorporate all motions faster than about 10-5-106 s, th echaracteristic time scale for the NMR measurement (Seelig& Seelig, 1974; Petersen & Chan, 1977). Thus, the contri-bution from any one of the possible motions (trans-gaucheisomerization, acyl-chain librations, rigid body m otions, etc.)to the spectrum is difficult to determine. Oth er commonlyused spectroscopies, primarily fluorescence and ESR, are ex-quisitely sensitive, but in general require the use of probemolecules. These at best present difficulties in transferringthe measured spectral properties of the probe to the physicalproperties of the phospholipid and at worst have the po tentialto perturb the order and dynamics of the system under in -vestigation (Taylor & Smith, 1980).Infrared and R aman spectroscopies, in principle, ope rateon a time scale that has the potential to sample directly the

0006-2960/89/0428-8934$01 SO10 0 1989 American Chemical Society


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Quantitative Determination of Bilayer Disorderfastest conformational alterations that occur in biologicalmem branes , namely acyl-chain trans-gauche isomerizations.Qua ntitative determination of this motion is desirable both forthe calibration of theoretical models for membrane structureand for data interpretation from other spectral techniqueswhere trans-gauche isomerization forms but one componentof the detected motion.To da te, vibrational spectroscopy has been used primarilyin a qualitative way to m onitor lipid-phase changes and theeffect of other mem bran e com ponents on them. Several widelyused indices of acy l-chain order are e xta nt-e .g., the 2850-cm-Ifrequency of the acyl-chain C-H stretching modes (Men-delsohn & Mantsch, 1986) from FT- IR spectra, the 2935/2880 and 2850/2880 intensity ratios from phospholipid Ramanspectra (Mush ayaka rara et al., 1982; Taraschi & Mendelsohn,1980), and the intensity of the 1130-cm-I band arising fromC-C stretching frequencies of the acyl chains in the transconformation (Gab er & Peticolas, 1977 ; Yellin & Levin, 1977;Lieb et al., 1982; Levin & Bush, 1982).Yet it has been difficult to correlate these empirical spectralparame ters with a quan titative description of trans-gaucheisomerization in the acyl chains. Several models have appearedfor the intensity of the 11 30 cm-I band in this context (G aber& Peticolas, 1977; Pink et al., 19 80). Thes e, in turn, have beencriticized on the grounds th at the mode is sensitive not onlyto trans-gauche isomerization but also to additional structura lreorganizations in bilayers (Snyder et al., 1980).Th e current study describes the successful measurement ofthe C D 2 rocking modes in the I R spec tra of specifically deu-terated phospholipids and their implementation as direct andquantitative probes of conformational order in phospholipidsthat have been deuterated at specific acyl-chain positions.Integrated band intensities have been incorporated in aquantitative model for the fraction of gauche rotame rs formedat particular positions (depths) in the bilayer. The methodwas originally proposed by Snyder and Poore (1973) as aspecific probe of hydrocarbon chain conformation in alkanesand later applied by Snyder, Strauss, and their co-workers(Maroncelli et al., 1985a,b; Shannon e t al., 1989) to monitorthe distribution of gauch e bonds in a variety of solid (rotator)phases in these systems. Th e current ap plicatio n of the methodis the first to the study of conformational disorder in phos-pholipid bilayers.MATERIALSN D METHODS

Syntheses of Specifically Deuterated Phospholipids.Syntheses of 4,4,4,4-d4-dipalmitoylphosphatidylcholine 4-d4-DPPC), 6,6,6,6-d4-dipalmitoylphosphatidylcholine 6-d4-DPPC), and 10 , l 0, lo, 10-d4-dipalmitoylphosphatidyl-choline (1O-d,-DPPC) were modifications (H. F. Schuster,unpublished results) of the procedures of Tulloch (1979 ) scaledto yield 2-3 g of each phospholipid. Th e various derivativeswere fully characterized by NMR, mass spectrometry, andFT-IR . Differential scanning calorimetry scans for each de-rivative displayed a sha rp (half-width 0.3 C ) main transitionat 41 OC and a pretransition at about 32 OC.Sam ple Preparation. Complete sample hydration is criticalto the success of this experiment. Incomplete hydration leadsto the appearance of extra FT-IR feautres of unknown originthroughout the C D2 rocking region (600-660 cm-I), vitiatingthe results. Such spurious bands were observed repeatedly innumerous attempts to measure the acyl-chain isomerizationprocess by use of attenuated total reflectance (ATR) tech-niques. Furthermore, ATR measureme nts were rendered moredifficult by the appe arance of interference fringes of greaterintensity than the sought spectral features.

Biochemistry, Vol. 28, No. 22 , 1989 8935Conditions for observations of CD 2 rocking modes wereoptimized in a transmission experiment. Ten milligrams ofspecifically deuterated lipid and 20 pL of D 2 0 were added toa culture tube, which was then sealed. Th e mixture was helda t 5 5 OC, with periodic agitation, for a t least 1 h, and usuallylonger, to ensure complete hydration. Hydra ted samples wereplaced between two AgCl windows, contained and sealed witha 6-pm spacer. Th e circumference of this assembly waswrapped with Teflon tape, forming a further seal againstdehydration. Th e assembly was then inserted into a varia-ble-temperature Harric k cell. Tem peratu re control wasachieved with a H aak e circulating water bath and monitoredwith a thermocouple placed adjacen t to the point where theIR radiation was focused. Temp eratures ar e estimated ac-curate to f l OC.FT-ZR Dat a Acquisition. Spectra (Rutge rs University) wereacquired with a Mattson Instruments Sirius 100 FT-IRspectrometer (Mattson Instruments, Madison, W I) equippedwith a HgCdTe detector (1 X 1 mm) fabricated (InfraredAssociates, Cranbury, N J) to give high sensitivity near 620cm-; 2000 scans of sample and background were co-addedand apodized with a triangular function. Spec tromete r reso-lution was 4 m-I. Interf erograms were zero-filled (two levels)

and fast Fourier transformed to yield data encoded every 1cm-l. Band positions, determined with a center of gravityalgorithm (Cameron et al., 1982), had an uncertainty of lessthan fO.l cm-I.A parallel series of spectra for each of the specificallydeuterated phospholipids were obtained on a Digilab FTS-40FT-IR spectrometer (Digilab, Inc., Cambridge, MA ) locatedat the B attelle laboratories. These IR spectra w ere collectedby co-addition of 1024 interferog rams at 2-cm-I resolution withone level of zero-filling and apodized with a triangula r fun ctionto give a final data encoding interva l of 1 cm-I. For theseexperiments, the spe ctrometer was equipped with a wide-band(-400 cm-I cutoff) Hg Cd Te detector.FT-IR Data Reduction. The CD2 rocking modes discussed

in this study app ear between 60 0 and 660 cm-I as very weakfeatures on a large D2 0 ibration background. Typically thissloping backg round is 0.8-1.2 absorb ance units under theexperim ental conditions described above. Background sub-traction is necessary because the strongest band of this spectralregion is about 30 milliabsorbance units in the liquid-crystalphase. Fully proteated DPPC , hydrated with molar propor-tions of D 2 0 dentical with those of the deuterated sam ple andmeasured under the same conditions of cell path length andtemperature, is used as the background for subtraction.Subtraction is completed when minima between 610 and 660cm-I have equal absorbance values.Incomplete resolution of spectral features made curve fittingfor quantitative analysis of band intensities necessary. Band

profiles constructed from addition of individual Gaussian-Lorentzian bands were compared with expe rimental spectraby using an intera ctive algorithm w ritten specifically for thispurpose (J. Brau ner, unpublished results). Area s of thecomponent bands were computed and used for calculating therelative intensities of various conformers.An alternative method of backgroun d subtraction was usedat the B attelle laboratories to visualize the weak CD, rockingmodes in the region 600-700 cm-. In this metho d, a mul-tipoint polynomial function was fit to the sloping backgroundin the region between 685 and 605 cm-. This functio n wasthen subtracted from the spectrum of the deu terated lipid togive a flat base line in this region. Curv e fitting of the resultingspectrum to obtain quantitative band intensities and are as was


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8936 Biochemistry, Vol. 28, N o. 22 , 1989Table I: Percent of Total" Gauche Conformers for SpecificallyDeuterated DPPC Derivatives

temp ("C)

Mendelsohn et al.

derivative 20 33 f 1 38 48.5 f 0.54-d4-DPPC 7.6 10.9 f .1 (4)* 1 1 . 1 f .8 (4) 20.7 & 4.2 (5)6-d4-DPPC 1.7 & 0.3 (4) 2.3 f 1 .1 (3) 32.3 f 2.3 (4)10-dd-DPPC 2.5 19.7 f 0.8 (2 )

"The totals neglect gg conformers. See text. *Nu mbe r of mea-surements i n parentheses.Table 11: Percent of tgt" and tgga Conformer Classes for SpecificallyDeuterated DPPC Derivatives at 48 OC

derivative tgt class (%) tgg classb (%)4-d4-DPPC 84.6 f 8. 9 (5 ) 15.4 f 8. 96-dd-DPPC 74.4 f 18.1 (5 ) 25.6 f 18.110-dd-DPPC 85.9 f 8.0 (2) 14.1 f 8.0

aConformer classes are defined in the text. *Determ ined by differ-ence.accomplished by using a least-squares algorithm (Fraser &Suzuki, 1973). The areas and intensities of the calculatedcomponent bands were used in the calculation of the relativeconformer percentages. The data given in Tables I and I1 werecalculated by using spectra recorded both at the Battellelaboratories and at Rutgers University. Good agreement wasobtained between the two methods described above forbackground subtraction, as reflected by the standard deviationsreported in Table I, which are based on all the data obtainedin both laboratories.T H E O R Y

Maroncelli et al. (1985a,b) have shown that the frequencyof a C Dz rocking mode substituted into a hydrocarbon chainis sensitive to conformation in the immediate neighborhoodof the CD , moiety. We follow their suggestion and italicizethe C-C bonds tha t are directly bonded to the CD,. Thus,t g means the C Dz group under study adjoins a trans (t) anda gauch e (g) bond. The C D2 probe frequency is dependenton the C-C bond or bonds neighboring the tt and tg bond pairs.I n particular, there are three discrete frequencies in th e620-660-cm-' spectral region tha t are relevant to the curr entwork. An IR-active band occurs at about 622 cm-' if the CD 2group adjoins two trans bonds, independent of the confor-mation of neighboring C-C bonds. This class of conformerswill be termed the tt class. A second band at about 652 cm-'consists of conformations of the type g'gtt (kink ) and ttg t(single gauche bend). The final band of interest for confor-mational study appea rs at 646 cm-l an d is assigned as the CD 2rocking modes arising from ttgg or g'tgg. Maroncelli et al.( 1 985a,b) have shown that the rocking frequencies are notsensitive to the conformation of C-C bonds more than twobonds away from the C D2 group. Conformers of the classgiving rise to the band at 6 52 cm-I are designated tgt, andthose of the class giving rise to the 646-cm-l feature aredesignated tgg. Taken tog ether, these two classes delineatethe extent of disorder at a given chain position. In all currentexperiments, the 646-cm-I band is much weaker than the652-cm-I band as expected from the natur e of the conformers.The ggconformations themselves are expected to have a weakbroad band calculated a t 680 cm-' but observed in cycloalkanes(Shann on et al., 1989) at 665 cm-l. These rare conformersare neglected in the current analysis.As trans-gauche isomerization occurs on a time scale slowerthan the vibrational period, the various conformations give riseto discrete (albeit weak) bands in the IR. The ratio of gauche(n,) to trans (n,) orms is given by

n, Z(652) +I(646)where the factor A allows for differences in unit absorptivitybetween the bands and Zexps the experimentally measuredintensity ratio.The factor A was determined (Maroncelli et al., 1985) froma statistical model of alkanes and a calculation of the bandintensities from the square of the dipole moment derivativesfor the particular norm al modes. An energ y difference of (508f 50) cal/mol was assumed for the relative energy of g vs t.This led to A = 1.00 f .07, the uncertainty reflecting therang e of experimental values for the energy difference. Th eindividual bands of the 652/646-cm-I doublet ar e also cal-culated to have equal intensities; thus, no further correc tionsto the numerator of eq 1 ar e required. The fraction of gaucherotamers is thus computed from

RESULTSTh e spectral regions of interest in the cu rrent work encom -pass the CD, stretching region (2000-2300 cm-l) and the CD 2

rocking modes (60- 66 0 cm-I). Typical data above and belowthe gel-liquid-crystal phase transition (41 "C) for thesespectral regions, respectively, are shown in part A and B ofFigure 1 for 6-d4-DPPC. These spectra have had solventbackground subtracted and have been flattened as describedunder Materials and Methods. The symmetric and asym-metric CD, stretching frequencies near 2100 and 2200 cm-I,respectively, have been used as qu alitative probes of the gel-liquid-crystal phase transition by Ram an spectroscopy (Bansilet al., 1980) and by IR spectroscopy (Cameron et al., 1981).To demonstrate tha t the current set of D PPC derivatives havethermotropic behavior resemb ling the fully proteated molecule,the frequency of the symm etric CD 2 stretching mode has beenmonitored as a function of temperature. Th e results are shownin Figure 2. Th e cooperative transition with T,,, at 41 OC isin good accord with the well-known gel - iquid-crystaltransition temperature for this molecule (Hinz & Sturtevant,1972). Thu s, it is evident tha t insertion of four deuterons doesnot significantly alter the thermotropic behavior of the mol-ecule and that hydration of the sample is complete under thecurr ent experimental conditions. Incomplete hydration isknown to significantly increase T , (Chapman, 1975).Th e conformationally sensitive CD 2 rocking modes for 6-d4-D PPC above an d below T , are shown in Figure 1B. Thebands are rather weak in intensity-the absorbance values (notshown in the figure) ran ge from 0.040 for the 622-cm-I tra nsband in the gel phase to less than 0.001 for the 652- or 646-cm-' gauche bands in the gel phase. The noise level after da tareduction is estimated a t 0.0001 unit. Th e complex contoursin this spectral region have been decomposed as discussedunder M aterials and Methods, and the resultant fractions oftotal gauche (tgt +tgg classes) conformers derived from t heintensities of the component bands for the 4, 6, and 10-d4-DPPC derivatives studied are listed in Table I. An exampleof the qu ality of the fit is given in Fig ure 3 for 6-d4- DPP Cabove T,. The spectral assignments for the bands at a bout622, 646, and 652 cm-I have been discussed above. Additionalweak features in the 610-630-cm-] region are of unknownorigin, but they contribute less than 3% of the intensity of themain 622-cm-I band and thus do not seriously affect theconform ational analysis below. A band near 661 cm-' isrelatively insensitive to confo rmational alterations and has been


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Qua ntitative Determ ination of Bilayer Disorder Biochemistry, Vol. 28 , No. 22 , 1989 8937

2155 2105 2055Frequency (cm.-1)

653 6 4 3 633 6 2 3 61 3F requency (cm.-11

FIGURE1: (A ) FT-IR spectra in the C-D (2055-2200 cm-I) stretchingregion for 6-d4-DPPC at the indicated tempera tures above (49 "C)and below (35 "C) T,. The frequency of the symmetric stretch near2100 cm-l is measured and used to construct a melting curve (Figure2) . (B ) FT-IR spectra in th e CD2 rocking region (610-665 cm-l)for 6-d4-DPPCat 33 (- -) an d 48 "C (-). The bands near 646 and653 cm-' arise from gauche conformers as described in the text. Theweak intensity in the gel phase implies a highly ordered system. Theband near 622 cm-' arises from trans conformers. These spectra havenot been subjected to spectral smoothing algorithms and reflect theraw data after background subtraction and base-line flattening. Theabsorbance of the 622-cm-l band at 48 OC is 18 milliabsorbance unitsabove the background.. %-- I0 1

uLw3e ?2x "

I x , ~ l x l , l Il 1 1 1 1 l 1 1 128 32 36 40 44 48

TE3P I C 1FIGURE 2: Melting curve for 6-d4-DPPC constructed from the fre-quency of the CD2symmetric stretching mode. The sharp transitionnear 41-42 "C s th e main phase transition.assigned by Maroncelli et al. (1985) as arising from CHDrocking modes of incompletely deute rated methylenes. It istherefore irrelevant to the current analysis. The results aredepicted in graphical form in Figure 4. In Table 11 , th erelative contributions of the individual tg t (652 cm-l) and tgg(646 cm-l) bands to th e liquid-crystlaline sta te of th e variousderivatives are summarized.DISCUSSION

As noted above, insertion of CD, groups at specific sites onthe phospholipid acyl chai ns provides a set of nonperturbing

654 644 634 624 614Frequency lcm.-11

FIGURE 3: Results of a typical curve-fitt ing process for band de-composition of the CD2rocking region. The experimental spectrumis the solid line (-). The dotted curves (-) are the component bands.The dashed line (-- -) represents th e sum of the component bands.

aa0.2

0 0.1a0.0

wI3

* - - - - - - - x

0 2 4 6 8 1 0 1 2POS IT IONFIGURE 4: Graphical summary of results showing disordering of theDPPC acyl chains as a function of depth in the bilayer. The errorbars for each data point are drawn in accord with the data in TableI . Temperatures are (0) 8.5, (A) 38, an d (X ) 33 OC. The horizontalline represents the theoretical maximum disorder for a random coila t 41 "C (Gruen, 1982).IR structu ral probes. The current experiment allows thefollowing issues to begin to be quantita tively addressed: (1)Wh at a re the fractions of trans and gauche conformers presentin the gel and liquid-crystal phases? (2) W hat is the depthdependence of the trans/gauche population ratio along the acylchains? (3 ) What types of conformers are present at eachposition? (4 ) How do the measurements of trans-gaucheisomerization compare with the order p arameters of Z HN M Rspectroscopy when the la tter a re analyzed to remove contri-butions from slower motions?The assumptions inherent in the analysis of these data areas follows: (1 ) The relative gauche/trans absorptivities (factorA in eq 1) are the same in phospholipids as in alkan es. Asthe vibrational modes a re highly localized (thu s not sensitiveto bulk phase struc ture), this assumption is well justified. (2)The vibrational bands are sensitive only to trans-gaucheisomerization. Th e rapid time scale should "uncouple" thesevibrational frequencies from the slower motions that appearin N M R .Several different experimental approaches have been usedto generate estimates of the extent of conformational orderin the phospholipid acyl chains. Yellin and Levin (1977), frommeasurements of the C-C stretching bands near 1090 and1130 cm-' in the Ram an spectrum , estimate the occurrenceof one gauche bond/acyl chain in the gel phase of DPPC.Gaber and Peticolas (1977) used the same 1130-cm-' featureto define an order parame ter r elated to the number of transbonds in the acyl chains. They conclude that 12 trans bondsexist/acyl chain in a dispersion of DPPC at 30 "C, two transbonds are lost between 34 and 41 'C (e.g., between the pre-transition and the main transition), and an additional fivebonds ar e lost at th e main transition. The basic assumption


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8938of their analysis is that the intensity of the 1130-cm-' modeis a consta nt per "trans-unit", independent of neighbor andend-group effects. Implicit also is th e idea tha t the 1130-cm-'intensity changes ar e derived solely from trans-gauche isom-erization. The latter assumption was challenged by Snyderet al. ( l98 0) , who showed tha t in n-heneicosanethe 1130-cm-' band decreased by 20% on transition of thismolecule from the orthorhombic to the hexagonal phase, atransition during which the all-trans conformation is preserved.

A different approach was proposed by Pink et al. (1980)in which the formalism of Theimer (1957) was used to cal-culate the l 130-cm-' intensity for particular conformations.Practical application of the model requires a theory of chainconformations in which the locations of gauche conformationscan be predicted. Th e model has been criticized by Vogel andJahnig (1981). The quan titative applications of vibrationalspectroscopy to date to the problem of conformational orderin phospholipids are thus quite confused. The basic difficultyarises because the 1130-cm-' mode has internal coordinatesfrom any section of the molecule that is in the trans config-uratio n. Th e presence of end effects and conformation ofneighboring groups, as suggested by Gaber and Peticolas(1977), and the a ppar ent sensitivity of the mode to struc turalchan ges not related to trans-gauche isomerization, cre atedifficulty with the detailed analysis.Other physical techniques have been applied to this problem.Nagle and Wilkinson (1978) measured the volume change atthe main transition, estimated the energy difference accom-panying this change, and deduced that the change in theprobability of a ga uche rotamer at the main transition [ Ap ,in the notation of Nagle an d Wilkinson (1978)] is 0.23. Theyhave not dealt with the position dependence of this probability.Their analysis leads to an estimate of 3.8 gauche rotamer s/chain above T , (7,6/molecule) for DPPC .Several estimates of intramolecular order in the acyl chainshave been made on the basis of NM R measurements. Seeligand Seelig (1974), in their pionee ring application of *H N M R ,

have analyzed the measured deuterium order parameters andrecognized a plateau of constant values from position 2 toposition 10 in the acyl chain of D PP C and decreased orderfrom position 10 to the termina l methyl region. By selectingparticularly likely conformations and analyzing th eir contri-butions to the order profiles, the Seeligs determined tha t thelower limit of gauche st ates per chain was three. To conformto the estimated energy difference of 500 cal/mol between tand g states, an upper limit of six gauche states per chain wasestimated. Petersen and Chan (1977) have elaborated atheoretical model invoking chain reorientation and chainisomerization via kink diffusion to explain proton and deu-terium order parameters. They calcula te a probability of transorientation of a methylene segment in the upper part of thechain to be about 0.8-0.9 in the liquid-crystalline state.The curr ent experimental determination of configurationalorder at particular chain positions can be directly comparedwith some of the abo ve results. Our observation of 20.7 f .2,32.3 f 2.3, and 19.7 f .8% gauche rotamers at the 4, 6, and10 positions, respectively, of DPPC at 48 OC is in fairagreement with Petersen and Chan's model. Within the limitsof current experimental precision (Table I) there is insignif-icant difference in gauche rotamer probability between pos-itions 4 and 10, although a real increase appears at the 6-position.To estimate the number of gauche rotamers/chain requiresmultiplication by the appro pria te number of C-C bonds,naturally excluding the C15-C ,6 bond. This results in 3.6

Biochemistry, Vol. 28, No. 22, 1989 Mendelsohn et al.gauche bonds/chain, assuming an average of our FT-IRmeasurem ents along the entire chain. It is noted this isprobably a lower bound to the true number, as significantlymore disorder is expected from C ,,-C I6, an area of th e bilayernot probed in the current work. The appropriate deuteratedderivatives of DP PC are currently being synthesized to evaluateconformational order toward the bilayer center. Assuminga maximum value of 36.8% [for a random coil at 41 OC(Gruen, 1982)] for bonds 11-15 yields a maximum numberof about 4.2 gauche bonds/chain. These results (lower limit=3.6; upper limit = 4.2) are in excellent accord with theanalysis of the dilatometric data by Nagle and Wilkinson(1978) and with the lower range of the estimate of the Seeligs(1 974) from NM R measuremen ts. Examination of Figure 4confirms tha t significant restrictions to m aximum disorde r existin the upper half of the chain compared to a random coil, forwhich, as noted above, 0.368 of the bonds a re expected to begauche.

Conformatio nal order in th e gel sta te at 33 OC is very highfor the 6- and 10-positions of the acyl chains, where only1. 53 % gauche bonds are found (Table I). The large fractionaluncertainties (Table I) reflect the low absorbance values ofthe gauche marker band (


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Quantitative Determination of Bilayer Disorderas defined under Theory, while the 652-cm-' band arises fromthe tg t class. W e note that kinks cannot be separated fromsingle gauche rotamers.The primary drawbacks to the current FT-IR experimentare th e requirements for specifically deuterated material andthe weakness of the IR absorption bands. Th e syntheses ofthese specifically deuterated PCs have been improved to thepoint where it is reasonable to generate 1-3 g of ma terial ( H.Schuster, S.S. Hall, and R. Mendelsohn, unpu blished results).The weakness of the IR bands is the limiting factor in theultimate utility of our approach. The ba nds (whose absorbancevalues at the path lengths used range between 0.040 and 0.001)sit on an intense background arising from solvent librationalmodes. Switching from H 2 0 o D,O reduces the interferenceto a level of about 1 0-1.5 absorbance units at the path leng thsutilized. Increasing the path length to increase the absorbanc eof the C D2 groups raises the underlying solvent absorbanceto an unacceptable level.With current technology, it should be possible to examinethe effect of small molecules such as steroids and peptides onthe lipid conformational order. Finally, the quantitative de-termination of acyl-chain order reported should be a usefultest for statistical m echanical theories of bilayer conform ation[e.g., Pink et al. (1982)].

Registry No. 4-d4-DPPC, 122844-70-6; 6-d4- DPPC , 122844-7 1-7;IO-dd-DPPC, 122844-72-8; DPPC, 2644-64-6.REFERENCESBansil, R., Day, J. , Meadows, M., Rice, D., & Oldfield, E.(1980) Biochemistry 19, 1938-1943.Cameron, D. G., Casal, H . L., Mantsch, H. H., Boulanger,Y. , & Smith, I. C. P. (1981) Biophys. J . 35 , 1-16.Cameron , D. G., Kaupinnen, J. K., Mo ffatt, D. J., & Mantsch,H. H . (1982) App l . Spectrosc. 36 (3) ,245-249.Chapman, D. (1975) in Cell Mem branes, Biochemistry, CellBiology and Pathology (Weissmann, G., & Claiborne, R.,Eds.) H P Publishing, New York.Fraser, R . D. B., & Suzuk i, E. (1973) in Physical Principlesand Tec hniques of Protein Chem istry (Leach, S . J., Ed.)Academic Press, New York.Gaber, B. P., & Peticolas, W. L. (1977) Biochim. Biophys.Acta 465, 260-274.Gruen, D. W. R. (1982) Chem. Phys. Lipids 30, 105-120.Hinz, H. J., & Sturtevant, J. M. (1972) J . Biol. Chem. 247,Levin, I. W., & Bush, S. F. (1981) Biochim. Biophys. Acta6071-6075.640, 760-766.

Biochemistry, Vol. 28, No. 22, 1989 8939Lieb, W. R., Kovalycsik, M., & Mendelsohn, R. (1982)Biochim. Biophys. Acta 688 , 388-398.Maroncelli , M., Strauss, H. L., & Snyder, R . G. (1985a) J.Chem. Phys. 82, 281 1-2824.Maroncelli , M., Strauss, H . L., & Snyder, R. G. (1985b) J .Phys. Chem. 89, 4390-4395.Mendelsohn, R., & Mantsch, H. H. (1986) in Progress inProtein-Lipid Inte raction 2. (Watts, A., & de Pont, J. J .H . H . M. , Eds.) Elsevier Science Publishers (BiomedicalDivision), Amsterdam .Mushayakarara, E., Albon, N., & Levin, I. W. (1982) Bio-chim. Biophys. Acta 686 , 153-159.Nagle, J. F., & Wilkinson, D. A. (1978) Biophys. J . 23 ,Pearson, R . H., & Pascher, I. (1979) Nature 281,499-501.Petersen, N. O., & Chan, S. I. (1977) Biochemistry 16,Pink, D. A., Green, T. J . , & Chapm an, D. (1 980) Biochem-istry 19, 349-356.Pink, D. A,, Lookman, T. , MacDonald, A. L., Zuckermann,M. J., & Jan, N . (1982) Biochim. Biophys. Acta 687,Seelig, A., & Seelig, J. (1 974) Biochemistry 13, 4839-4845.Seelig, J ., & MacDonald, P. M. (1987) Acc. Chem. Res. 20,Shannon, V. L., Strauss, H . L., Snyder, R . G., Elliger, C. A.,& Mattice, W. L. (1989) J . Am. Chem. SOC.111,Snyder, R . G ., & Poore, M. W . (1973) Macromolecules 6,Snyder, R. G., Cameron, D. G., Casal, H . L., Compton, D.A. C. , & Mantsch, H. H. (1980) in Proceedings VZIthInternational Conference on Ram an Spectroscopy (Murphy,W. F., Ed.) North Holland Publishing Co., Amsterdam.Taraschi, T., & Mendelsohn,R. (1980) Proc. Natl. A cad. Sci.Taylor, M. O.,& Smith, I. C. P. (1980) Biochim. Biophys.Theimer, 0 .H. (1957) J . Chem. Phys. 27, 408-416.Tulloch, A . P. (1979) Chem. Phys. Lipids 24, 391-406.Vogel, H., & Jahnig, F. (1981) Chem. Phys. Lipids 29,Westerman, P. W., Vaz, M. J., Strenk, L. M., & Doane, J.W. (1982) Proc. Na t l . Acad. Sci . U .S .A .7 9 , 2890-2894.Wittebort, R. J., Olejniczak, E. T., & Griffin, R. G . (1987)J . Chem. Phys. 86, 5411-5420.Yellin, N., & Levin, I. W. (1977) Biochemistry 16, 642-647.

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U. S . A .77 , 2362-2366.Acta 59, 140-149.

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quantitative determination of conformational disorder in the acyl chains of phospholipid bilayers by...

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