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Bioscience Reports Vol. 6 N o. 3 1986

R e v i e w

I n fr ar e d S p e c tr os c op i c S tu d i e s o fi o m e m b r a n e s a n d M o d e l M e m b r a n e s

D a v i d C L e e a nd D e n n i s C h a p m a nReceivedKEY W OR DS: IR; mem branes; protein structure; lipid phase transitions; lipid-protein interactions

I N T R O D U C T I O NT h e u s e o f v i b r a t i o n a l s p e c t r o s c o p y to p r o b e t h e s t r u c t u r e a n d d y n a m i c s o f

b i o l o g i c a l m o l e c u l e s i s a r a p i d l y e x p a n d i n g a r e a o f r es e a r c h . I t s o r i g i n s , h o w e v e r , l ie i nt h e e a r l y s tu d i e s o f s i m p l e , o r g a n i c m o l e c u l e s b y i n f r a r e d a n d R a m a n t e c h n i q u e s . Th ec o m p a r i s o n o f v i b r a t i o n a l s p e c t ra w i t h m o l e c u l a r s t r u c t u re p r o v i d e d o r g a n i c c h e m i s tsw i t h c o r r e l a t i o n c h a r t s w h i c h a r e t h e b a s is o f sa m p l e a n a l y si s t o d a y . A s t a n d a r d t e x td e s c r i b i n g t h i s a p p r o a c h i s t h a t o f Be l l a m y 1 97 5) . A n a l y s i s o f b i o l o g i c a l m o l e c u l e s i s am o r e r e c e n t d e v e l o p m e n t a n d e x c e l le n t r e v ie w s a r e t h o s e o f S u s i 1 9 6 9) , F r a s e r a n dM a c R a e 1 97 3) , F a w c e t t a n d L o n g 1 97 3) , a n d T h o m a s a n d K y o g o k u 1 97 7) . T h ea p p l i c a t io n o f i n f r a re d a n d R a m a n t e c h n iq u e s i n m e m b r a n e r e s e a r c h h a s b e e nr e v ie w e d b y W a l l a c h e t a l . 1 97 9), F r i n g e l i a n d G u n t h a r d 1 98 1), A m e y a n d C h a p m a n1 98 3) , Le v i n 1 98 4) , a n d C a s a l a n d M a n t s c h 1 98 4) . Th e a m o u n t o f s t r u c t u r a l a n d

f u n c t i o n a l i n f o r m a t i o n a v a i l a b l e t o t h e I R s p e c t r o s c o p i s t w h o i s i n t e r e s t e d i n b i o l o g i c a ls y s t e m s is e x p a n d i n g r a p i d l y w i t h t h e a p p l i c a t i o n o f i n c r e a s i n g l y s o p h i s t i c a t e dm e t h o d s f o r d a t a a c q u i s i t i o n a n d a n a l y s i s a n d n e w t e c h n i q u e s i n s a m p l e h a n d l i n g .T h e s e t e c h n i q u e s i n c l u d e th e d e t e c t i o n a n d a s s i g n m e n t o f m i n o r s p e c tr a l c o m p o n e n t su s i n g d e r i v a ti v e a n d d e c o n v o l u t i o n c a l c u l a ti o n s , t h e s t u d y o f b io c h e m i c a l r e a c t i o n su s i n g k i n e t i c I R s p e c t r o s c o p y a n d t h e u s e o f c y l i n d r i c a l i n t e r n a l r e f l e c t i o n ce ll s f o r e a s ya n a l y s i s o f a q u e o u s s a m p l e s .

I n i t i a l l y , t h e m a j o r p r o b l e m i n t h e s t u d y o f b i o l o g i c a l m o l e c u l e s w a s t h ea b s o r p t i o n o f l i q u id w a t e r o v e r m u c h o f t h e I R s p e c tr u m . T h i s s e v e re l y l i m i te d t h ea n a l y s i s o f s a m p l e s i n t h e i r n a t u r a l s t a t e a n d n e c e s s i t a t e d t h e u s e o f h i g hc o n c e n t r a t i o n s , l o w p a t h l e n g t h c el ls l es s t h a n 5 0 g m ) o r d e u t e r i u m o x i d e a s a s o l v e n t.T h e a d v e n t o f m i c r o p r o c e s s o r - c o n t ro l l e d s p e c t r o s c o p y h a s p e r m i t t e d t h e s u b t r a c t i o nDepartment of Biochemistry and Chem istry, Royal F ree Hospital School of Medicine University ofLondon), Row land Hill Street, London NW3 2PF.

2350144-8463/86/0300-0235 $05.00/0 9 1986 Plenum Publishing orporation

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236 Lee and Chapmanof background water absorptions from dilute samples (Cameron e t a l . 1979; Chapmane t a l . 1980) thereby enabling accurate assessment of native structures. This approachhas revolutionized the application of IR spectroscopy to the study of membranesystems in recent years.Vibrational spectroscopy has several advantages for membrane studies. Firstly,variations in frequency, linewidth, and intensity are sensitive to structural transitions ofboth lipid and protein components. Secondly, the vibrations of individual groupsprovide structural information on highly localized regions of the bilayer. Thus, C- -Hstretching absorptions of the lipid acyl chains are readily distinguished from thecarbonyl stretchings of the interracial region and the phosphate stretchings of the polarheadgroup. Of particular importance in the study of lipid-protein interactions is thenon-perturbing nature of the technique. The addition of an external probe molecule isnot required and the absorptions of the lipid and protein groupings reflect their genuineenvironments. Other techniques, such as ESR and fluorescence, are limited by theperturbations which the added reporter groups may induce. Finally, the time-scale ofthe molecular vibrations is of the order of 1013 s- 1 which ideally complements the ESRand NMR timescales of 108 s- a and 105 s- 1.

INSTRUMENTATIONThe application of the tradit ional, dispersive IR instrument in the biological field

expanded with the widespread use of minicomputers. Computer aquisition of IR dataallows the operator to store spectra in undegraded form. Spectral subtractions,enhancements and expansions may then be carried out at a later date (Chapman e t a l .1980). Following the advent of the fast Fourier transform algorithm, spectrometersbased on the Michelson interferometer are now widely used (Fourier transforminfrared). An FT- IR spectrometer is comprised of two parts: an optical benchcontaining an interferometer and a computer which controls all aspects of spectralscanning and analysis. The interferogram of a scan, or the sum of many such scans, isconverted by means of a fast Fourier transform into the conventional form oftransmit tance (or absorbance) versus wavenumber spectrum. A short description of themathematical treatments involved is that given by Griffiths (1980).The principal advantage of FT- IR compared with dispersive IR is that the formeris able to obtain spectra of higher signal to noise ratio in a given scanning time. Thisarises through the multiplex or Fellgett s advantage, the detector examines all of thescanned spectrum almost simultaneously, and the Jacquinot advantage, the highoptical throughput of the interferometer owing to the absence of slits. A furtheradvantage of FT-IR is the much greater abscissa (wavenumber) accuracy which isachieved v i a laser referencing.

Kinet ic nfrared Spe ctrosc opyKinetic IR spectroscopy (KIS) is a new technique which has been used toinvestigate the molecular basis of biological trigger processes. The method wasdeveloped by Kreutz and co-workers and a recent review which gives a brief

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IR Studies of Membranes 237description of the apparatus and its applications is that of Kreutz e t a l . 1984). Theseapplications include the visual transduct ion process where the absorption of lightinduces molecular changes in the chromophore, retinal, which are transferred to theprotein Siebert and Mantele, 1980). The light-induced reactions of bacteriorhodopsinin the purple membrane of H a l o b a c t e r i u m h a l o b i u r n have also been studied Siebert e ta l . 1982; Engelhard e t a l . 1985). In both cases the molecular changes involved takeplace on a time-scale too fast for conventional IR methods. In addition, the extent ofband overlap prevents the resolution of the band-shifts in the normal static IRspectrum.

The methods used for sample preparation are determined by the requirement forboth high sample concentration and the use of hydrated systems. A thin-film is formedon the IR crystal by dehydration from aqueous solvent and the film rehydrated witheither H20 or ZH20 by evaporation from saturated salt solution. The results obtainedby this technique, particularly with respect to bacteriorhodopsin, will be reviewed later.Although these workers note that many biological samples will not tolerate drying,little attent ion has been paid to the possibility of irreversible structural transitionsoccurring in these samples.

THE APPLICATION OF IR SPECTROSCOPY TO MEMBRANESYSTEMSestrict ions

The complexity of most biological molecules leads to difficulties in theinterpretation of their IR spectra. This arises from the possibility of 3n-6 normalvibrations where n is the number of atoms); a number which may be increased byovertones, combination tones, and band splitting. Often, unambiguous assignmentsare impossible because of the extent of band overlap and the variety of molecularmotions.

Biological membranes are in a liquid crystalline state at ambient temperatures.The lack of order in the hydrocarbon chains leads to a broadening of the IRabsorptions and a reduction in polarisation.As mentioned in the introduction, liquid water absorbs strongly over much of the

mid-infrared which has meant that many IR studies of biological materials have beenperformed using dry samples. The advent of microprocessor-controlled IR has allowedus to study membranes in their natural aqueous state, although the use of lowpathlength cells is required to prevent total loss of transmission. This in itself may leadto errors in the estimation of absorption coefficients, and the use of high sampleconcentrations may induce molecular association.

An alternative approach is the use of 2H20 whose absorptions do not obscure theamide I and II bands of proteins, as a solvent. However, under these conditionsvariations in H--ZH exchange, which is both temperature- and conformation-dependent may give rise to ambiguous band shifts.A further problem which may be experienced is the absorption of atmosphericwater vapour which overlaps with the amide I and amide II bands of proteins and may

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238 Lee and Chapmanc o n f u s e c o n f o r m a t i o n a t a s s i g n m e n t s , a s h a s re c e n t l y b e e n n o t e d b y u s L e e e t a I .1 98 5b ). T o c o m b a t t h is , t h e s p e c t r o m e t e r s h o u l d b e t h o r o u g h l y p u r g e d w i t h d r y g a s a iro r n i t r o g e n ) o r e v a c u a t e d d u r i n g s c a n n i n g .

hospholipidsS t u d ie s o f t h e I R s p e c t r a o f p h o s p h o l i p i d s h a v e p r o v i d e d m u c h i n f o r m a t i o n o n t h e

s t r u c t u r e o f th e a c y l c h ai n , in t e rr a c ia l a n d h e a d g r o u p r e g i o n s o f m e m b r a n e b i l a y e r sC a s a l a n d M a n t s c h , 1 9 8 4 ) . C o m p r e h e n s i v e t a b l e s d e s c r i b i n g t h e v i b r a t i o n a l

a b s o r p t i o n s o f p h o s p h o l i p i d s a r e i n c l u d e d in t h e re v i ew s b y F r in g e l i a n d G u n t h a r d1 9 81 ) a n d A m e y a n d C h a p m a n 1 98 3). T h e t y p e s o f n o r m a l v i b r a t i o n f o r a C H 2 g r o u p

a s p a r t o f a n e x t e n d e d p o l y m e t h y l e n e c h a i n a r e i l l u s t ra t e d i n F i g . 1 .

s y m m e t r i cs t r e t h i n g

ymmetrics t r e t h i n g

S c i s s o r i n g

W ~ i g g i n g

T w i s t i n g

R o c k i n gFig. 1. Ty pesof normal vibration. The infrared-active vibrations of a CH z group.

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IR Studies of Membranes 239T h e A c y l C h a i n R e g i o n

An early study of films of phosphatidylcholine by Chapman et al. 0967) providedassignments of the C--H stretching and bending modes, the carbonyl band, and avariety of phosphate vibrations. These results showed tha t the spectra, particularly inthe region below 1400cm -1, were remarkably dependent on temperature and themethod of sample preparation. Considerable fine structure was observed in spectrarecorded of samples after dehydration or at low temperatures. The band progressiondue t o C H wagging modes and the C H rocking band near 720 cm- 1 were found to beparticularly sensitive. For anhydrous samples, the single 720 cm-1 band present atroom temperature split into several components at - 186~ This transition coincideswith a change in acyl chain packing from hexagonal to orthorhombic according to X-ray diffraction of the same samples.

This type of study was extended by Fookson and Wallach 1978) to include films ofphosphatidylethanolamine and mixed films of phosphatidylcholine/phosphatidyl-ethanolamine 1:1). Phosphatidylethanolamine showed splitting of the CH2rocking fundamental even at room temperature indicating a orthorhombic ratherthan hexagonal chain packing. On monohydration, these samples showed a completeabsence of fine structure because of the decreased intermolecular interactions whichoccur as water is inserted into the headgroup lattice.The sharp main endothermic phase transition of aqueous phospholipid bilayersresults in pronounced alterations in the methylene band parameters Asher and Levin,1977; Cameron and Mantsch, 1978; Cortijo and Chapman, 1981). The band maximumfrequencies of the CH 2 asymmetric and symmetric stretching bands are sensitive to thestatic order of the acyl chains. The introduction of an increased proport ion of 9 a u c h econformers above the phase transition causes a shift in these bands to higherfrequencies see Fig. 2). The width of the IR absorptions is determined by rotational,translational and/or collisional effects Casal et al . 1980). Thus, the C H bandwidthsare sensitive to the degree of motional freedom of the CH 2 groups. They are sensitive,therefore, to the phase transition, but they also reflect changes which do not result in analteration in the proportion of 9 a u c h e conformers, such as the extent of librat ional ortorsional motion.

A study of the C H scissoring mode in hydrated bilayers ofdipalmitoylphosphatidylcholine DPPC) in the gel phase has shown that this band issensitive to the orthorhombic-like to hexagonal packing transit ion which occurs as thetemperature is increased towards the pretransition Cameron et al. 1980a). Thehexagonal phase gives a single band at 1468 cm -~ whereas the low-temperatureorthorhombic phase gives two bands near 1475 cm- 1 and 1465 cm- 1. In a separatestudy Cameron et al . 1980b), these workers demonstrated that changes in the gel-phase spectrum of DPPC, particularly those associated with the pretransition near37~ reflect alterations in interchain interactions and the packing of the fully extendeda l l - t ra n s acyl chains. Above 38~ the spectra reflect the hexagonal chain packing with ahigh degree of molecular motion. Below 36~ the spectra are sensitive to a reduction inaxial motion and a gradual introduction of orthorhombic packing.As part of their extensive studies of lipid phase behaviour by FT-IR, Mantsch et al.1982, 1983) have also studied the C--H modes of aqueous dispersions of

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240 Lee and Chapman

2924

2923

2922

m 2921Z< 292

2919

2918I0 2 3

T E M P E R A T U R E

I I4 O

~Fig. 2. The variation of the CH 2 asymmetric stretching frequencyof dimyristoyl phosphatidy lcholine bilayers with temperature.Band frequencies were measured from difference spectra generatedby the subtraction of the spectrum of the buffer at eachtemperature. The mid point of the phase transition occurs at 23 ~in agreement with calorimetric measurements.

p h o s p h a t i d y l s u lp h o c h o l i n e s a n d p h o s p h a t i d y l e t h a n o l a mi n e s . O n p a s s i n g t h r o u g h t h ema i n p h a s e t r a n si t io n t h e C H 2 s y mm e t r ic s t re t c hi n g b a n d o f d i p a l mi t o y l p h o s p h a t i d y l -e thano lam ine DP PE ) b i laye rs show s a la rge r inc rease in f requency com pare d wi th thecor respon d ing m ode fo r D P P C b i laye rs Casa l and M antsch , 1983). Th is sugges t s tha tm o r e g a u c h e b o n d s a r e i n t r o d u c e d a t t h e p h a s e t r a n s i t i o n f o r D P P E t h a n D P P C .Phos pha t idy le tha no lam ines wi th unsa tu ra ted acy l cha ins d i sp lay a reve rs ib le l iqu id -c rys ta ll ine to a non- lam el la r inve r ted hexagona l Hn) phase t r ans i t ion on fu r the rhea t ing above the phase t r ans i t ion t em pera tu re Tm L uza t t i et al. 1968). The t ransi t ioninvolves a fur ther increase in the g a u c h e conce n t ra t ion o f the l iqu id -c rys ta l l ine p hase a sr e v e al e d b y t h e t e mp e r a t u r e - d e p e n d e n c e o f th e C H z s y mm e t r ic s t re t c h in g f r e q u e n c y o fe g g y o l k P E Ma n t s c h et a l . 1981).

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IR Studies of Membranes 241The C- -H modes have also been used to investigate the phase behaviour of bovine

brain phosphatidylserine (PS) and its interaction with calcium ions (Dluhy et a l .1983a). As for PC, an increase in the CH 2 symmetric stretching frequency coincideswith the main phase transition. On addition of C a 2 , the acyl chains are furtherordered below T,, and a small monotonic increase in g a u c h e conformers occurs withtemperature--the phase transition being abolished.

T h e I n te r f a c ia l a n d H e a d g r o u p R e g i o n sThe structure of the interfacial region of lipid assemblies may be examined v i a the

ester group vibrations. The most intense of these bands are the C-~-O stretchingfrequencies between 1750 cm- 1 and 1700 cm- 1 Two absorption bands in this regionare associated with the two ester groupings in diacyl lipids. The s n - 1 carbonyl gives riseto a band near 1740 cm- 1 and a band near 1725 cm- 1 is associated with the carbonylat the s n - 2 position (Bush et al. 1980; Mushayakarara and Levin, 1980; Levin et a l .1982). This split ting arises, in part, because of the conformational inequivalence aboutthe C1-C 2 bonds of the s n - 1 and s n - 2 chains which adopt t r a n s and g a u c h econformations, respectively, and through possible differences in the extent ofhydration. Difference IR spectra of hydrated samples rlormally reveal only a singlebroad C ~ O band contour. The application of either spectral deconvolution (Casaland Mantsch, 1984) or second-derivative (Lee et a l . 1985b) calculations usuaUy revealstwo or more components.The midpoint of the broad C ~ O stretching band shifts by 2 cm-1 to higherfrequencies at the pretransition temperature; at the main transition there is a shift backto lower frequencies by 4 cm - 1 (Casal and Mantsch, 1984). These changes arise fromalterations in the relative intensity rather than shifts in the s n - 1 and s n - 2 components.The shift in the C~ O bands at the pretransition temperature is a reflection of a decreasein the angle of tilt of the acyl chains with respect to the bilayer normal. The lamellarliquid-crystalline to inverted hexagonal phase transition of unsaturatedphosphatidylethanolamines results in an increase in the C---~O stretching bandfrequency such that it is approximately the same as in the gel phase (Mantsch et a l .1981).The phosphate moiety of the headgroup gives rise to several strong vibrations inthe infrared. Asymmetric and symmetric stretching modes for the PO 2 group are foundnear 1250 cm - * and 1085 cm- ~, respectively (Casal and Mantsch, 1984; Arrondo et a l .1984). Weaker single bond P r O stretching modes are found in the region 900-800cm-* (Casal and Mantsch, 1984). A shoulder near 1060cm -~ on the PO2symmetric stretching band in the spectrum of DPPC is attributed to aR- -O - -P -- O- -R stretching mode (Arrondo et a l . 1984).Akutsu et a l . (1975) studied ordered films of DPPE by polarized IR spectroscopy.They found that the moments of the ~O stretching and the PO 2 and C---C--N +stretching modes are oriented almost parallel with each other and deviate by less than20 ~ from the plane of the film. The frequencies of the PO~- stretching =lodes aresensitive to the state of hydration of phospholipid bilayers. Dehydration results inband-shifts towards higher wavenumbers (Arrondo e t a l . 1984). These workers also

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242 Lee and Chapmanfound that these band-frequencies were insensitive to the main phase transition ofDPPC.

Phosphol ip id Cholesterol InteractionsThere have been several IR studies on the effect of the incorporat ion of cholesterol

on the static order of the acyl chains of aqueous phospholipids Asher and Levin, 1977;Umemura e t a l . , 1980; Cortijo and Chapman, 1981 ; Cortijo e t a l . , 1982). These studieshave shown that cholesterol causes an increase in the proportion of g a u c h e conformersbelow the phase transition temperature T,,) and a decrease in these conformers aboveT m compared with a pure lipid bilayer. This is manifested as an increase in both the CH2asymmetric and symmetric stretching frequencies below Tm and a decrease in theseparameters above Tm Cortijo and Chapman, 1981 ; Cortijo e t a l . , 1982). The width ofthe phospholipid phase transition, as detected by 1R, is increased by the presence ofcholesterol in the bilayer but the midpoint of the transition is unaffected Asher andLevin, 1977). At very high cholesterol concentrations e.g., 1.5:1 phospholipid:cholesterol molar ratio) almost no change in the relative proportions of t r a n s andg a u c h e conformers occurs with temperature Cortijo e t a l . , 1982). These observationsare in accord with the results obtained by calorimetric, NMR and ESR techniques.There is little IR evidence concerning the perturbation of the interfacial andheadgroup regions which may be exerted by cholesterol. A combined IR and Ramaninvestigation of the C= O stretching modes of DPPC Bush e t a l . , 1980) demonstratedthat no hydrogen bonding occurs between the fatty acyl carbonyl and the 3/~-OH groupof cholesterol in anhydrous samples. In hydrated samples, cholesterol reduces theconformational inequivalence between the s n - 1 and s n - 2 carbonyls by perturbing thelatter. This was revealed as a decrease in the relative intensity in the 1720 cm- 1 region.A study of the PO~- stretching modes and the asymmetric N CH3)3 stretching mode offully hydrated DPPC showed no variation in frequency on the introduction ofcholesterol into the bilayers Umemura e t a l . , 1980). This suggests that there is littleinteraction between cholesterol and the phosphorylcholine group.

Lip id Prote in In terac tion sIR spectroscopy is an ideal method for examining the perturbations of the lipid

bilayer which are introduced by the presence of integral polypeptides and proteins. I t isa technique which operates on a time scale complementary to the widely used NMRand ESR approaches and which does not require the addition of a reporter group to thesystem under study.Perturbations to the hydrophobic acyl chain region may be studied v i a the C--Hasymmetric and symmetric stretching frequencies. These parameters are sensitive tothe static order t r a n s / o a u c h e isomerisation) of the acyl chains. Corti jo e t a l . 1982) havereported the effects of incorporation of the intrinsic proteins Ca 2+-ATPase andbacteriorhodopsin, and the intrinsic polypeptide gramicidin A on the acyl chain orderof dimyristoylphosphatidylcholine DMPC) and DPPC bilayers. They found thatbelow Tm, these molecules behaved in a similar manner to cholesterol; that is theycaused an increase in the proportion of g a u c h e isomers. The presence of the intrinsic

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IR Studies of Mem branes 243m o l e c u l e s w i t h i n t h e b i l a y e r p r e v e n t s t h e a c y l c h a i n s f r o m a t t a i n i n g t h e a l l - t r a n sc o n f o r m a t i o n a t l o w t e m p e r a t u r e s . A b o v e T ,,, t h e p e r t u r b a t i o n o f t h e a c y l c h a i n s d i ff e rsf r o m t h a t o b s e r v e d w i t h c h o l e s te r o l . A t h i g h l ip i d : p r o t e i n m o l a r r a t i o s e .g .,D M P C / C a 2 + - A T P a s e o f 1 50 :1 ) a r e d u c t i o n in t h e p r o p o r t i o n o f 9 a u c h e c o n f o r m e r sw i t h r e s p e c t to t h e p u r e l i p i d b i l a y e r w a s o b s e r v e d . H o w e v e r , w h e n t h e c o n c e n t r a t i o n o ft h e i n t r in s i c p r o t e i n o r p o l y p e p t i d e w a s i n c r e a s e d , th i s e f fe c t w a s r e m o v e d a n d t h e s t a t ico r d e r o f t h e a c y l c h a i n s w a s e s s e n t i a ll y t h e s a m e a s in t h e p u r e l i p i d s y s te m . T h i s I Rs t u d y a l so p r o v i d e d f u r t h e r e v i d e n c e f o r a r e d u c t i o n i n t h e c o o p e r a t i v i t y o f t h e g e l t ol i q u i d - c r y s t a l l i n e p h a s e t r a n s i t i o n i n t h e p r e s e n c e o f t h e s e i n t r in s i c m o l e c u l e s . A t h i g hp o l y p e p t i d e / p r o t e i n c o n c e n t r a t i o n s a r e d u c t i o n i n T ,, w a s o b s e r v e d .

A q u a l i f ic a t i o n o f t h is t y p e o f s t u d y m u s t b e m a d e b e c a u s e o f t h e p o s s i b i li ty o fp r o t e i n a b s o r p t i o n s i n t h e s p e c t r a l re g i o n s t u d i e d . T h e p r e s e n c e o f h i g h i n t ri n s icp r o t e i n c o n c e n t r a t i o n s w i t h i n t h e l i p i d b i l a y e r s t r u c t u r e i n t r o d u c e s c o n s i d e r a b l ea m i n o a c id s i de - c ha i n c o n t r i b u t i o n t o t h e C - - H b a n d s . M o r e r e c en t ly , w e h a v ei n v e s t i g a t e d l i p i d - p r o t e i n i n t e r a c t i o n s u s i n g d i p e r d e u t e r i o m y r i s t o y l p h o s p h a t i d y l -c h o li n e, an a n a l o g u e o f D M P C i n w h i c h t h e ac y l c h a i n h y d r o g e n a t o m s a r e c o m p l e t e l ys u b s t it u t ed b y d e u t e r i u m a t o m s L e e et a l . 1 9 8 4 ) . G r a m i c i d i n A , a l a m e t h i c i n a n db a c t e r i o r h o d o p s i n , a s w e ll a s c h o l e s t e ro l , w e r e r e c o n s t i t u t e d i n b il a y e r s f o r m e d f r o mt h is l i pi d , e n a b l i n g a n i n v e s t i g a t i o n o f th e C - 2 H s t r e tc h i n g f r e q u e n c i e s w i t h o u ti n t e r f e r e n c e f r o m a m i n o a c i d s id e - c h a in s a t h i g h p r o t e i n c o n c e n t r a t i o n s . B a n df r e q u e n c y w a s u s e d a s a m e a s u r e o f t h e s t a t ic o r d e r o f t h e l i p id c h a i n s , w h i le b a n d w i d t h sg a v e i n f o r m a t i o n o n t h e l i b r a t i o n a l m o t i o n o f t h e c h a i ns . I t w a s s h o w n t h a t a b o v e t h el ip i d p h a s e t r a n s i t i o n t e m p e r a t u r e , l o w c o n c e n t r a t i o n s o f g r a m i c i d i n A a n da l a m e t h i c i n c a u s e d a s m a l l o r d e r i n g o f t h e l ip i d c h a i n s w h i l e b a c t e r i o r h o d o p s i n h a d n oe ff ec t. A t h i g h c o n c e n t r a t i o n s e a c h i n t r in s i c m o l e c u l e c a u s e d a d i s o r d e r i n g o f th e l ip i dc h a i n s a b o v e Tm. B a c t e r i o r h o d o p s i n h a d n o e ff ec t, a t e i t h e r c o n c e n t r a t i o n s t u d i e d , o nt h e r a t e o f a c y l c h a i n m o t i o n a b o v e Tm . B e l o w T , , , e a c h i n t r i n s i c m o l e c u l e c a u s e d ad i s o r d e r i n g o f t h e c h a in s a n d a n i n c r e a s e i n c h a i n m o t i o n c o m p a r e d t o t h e p u r e l i p idb i l a y e r .

W e p r e se n t, in F ig . 3, F T - I R s p e c t ra o f 2 -d 27 D M P C , a n a n a l o g u e o f D M P C i nw h i c h t h e a c y l c h a i n h y d r o g e n s o f t h e s n - 2 c h a i n h a v e b e e n r e p l a c e d b y d e u t e r i u ma t o m s b u t t h e s n - 1 c h a i n r e m a i n s u n m o d i f i e d . T h i s a ll o w s u s t o m o n i t o r s e p a r a t e l y t h ei n t r o d u c t i o n o f o a u c h e c o n f o r m e r s i n e a c h c h a i n v i a m e a s u r e m e n t o f t he C - - H a n dC - - 2 H s t r e t c h i n g f r e q u e n c ie s . A t 8 ~ w e l l b e l o w th e p h a s e t r a n s i t i o n t e m p e r a t u r e , t h eC H 2 a sy m m e t r ic a n d s y m m e t r i c s t r e t ch i n g b a n d s a r e f o u n d a t 2 9 2 0 c r n - 1 a n d2 851 c m - 1 a n d t h e c o r r e s p o n d i n g C H m o d e s a t 2 1 9 4 c m - ~ a n d 2 0 9 0 c m - ~ . A b o v et h e p h a s e t r a n s i t i o n i.e ., a t 3 6 ~ t h e C H z b a n d s a r e f o u n d a t 2 9 2 4 c m - t a n d2 8 54 c m - ~ a n d t h e C H a n d s a r e a t 2 1 9 6 c m - ~ a n d 2 0 95 c m - ~. S h i ft s i n th e s e b a n d sm a y b e u s e d t o e x a m i n e s p e c if ic p e r t u r b a t i o n s t o t h e s n - 1 a n d s n - 2 c h a i n s i n d u c e d b yt h e p r e s e n c e o f c h o l e s t e r o l, s i m p l e p o l y p e p t i d e s a n d m e m b r a n e p r o t e i n s i np h o s p h o l i p i d b i l a y e r s .

M a n t s c h a n d c o - w o r k e r s h a v e i n v es t ig a t e d t h e e ff ec ts o f t h e h u m a n e r y t h r o c y t em e m b r a n e p r o t e i n g l y c o p h o r i n o n th e I R s p e c tr a of D M P C b i la y e r s M e n d e l s o h n e tal . 1 9 8 1, 1 9 8 2 ; D l u h y e t a l . 1 98 3b ). T h e r e w a s n o I R e v i d e n c e f o r a n i m m o b i l i z e d l ip i da n n u l a r l ip i d ) c o m p o n e n t i n t h e s e m i x t u re s . T h e e f f ec ts o n t h e s t a ti c o r d e r o f t h e a c y l

c h a i n s w e r e e s s e n t ia l ly t h e s a m e a s t h o s e o b s e r v e d b y C o r t i j o e t al. 1 9 8 2 ) t b r C a 2 + -

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Lee and C hapman

Cbu~

2 4 4

3000 2750 2500 2250 2000WAVENUMBER (r I )

F i g 3 FT-IR spec tra of 2-d27 dimyristoylphosphatidylcholine DM PC) inwater a fter subtraction of the background water spectrum recorded at 8~solid line)and 36~ broken ine).Spectrawere recordedusing a Perkin-Elmermodel 1750 spectrometerat 4 cm -1 resolution and apodised with a mediumNo rton-Bee r function.A T P a s e a n d b a c t e r io r h o d o p s i n . T h e s e w o r k e r s a l so s t u d ie d t h e t e m p e r a t u r e -d e p e n d e n c e o f th e b a n d w i d t h o f t h e CH s y m m e t r i c s t r e tc h i n g b a n d . T h e p r e s e n c e o fg l y c o p h o r i n c a u s e d a n i n c r e a s e i n c h a i n m o t i o n s a t a ll t e m p e r a t u r e s s t u d i e d . As u b s e q u e n t s t u d y i n v e s t i g a t e d t h e t e r n a r y s y s t e m D P P C / D M P C - d 5 4 / g l y c o p h o r i n

D l u h y e t a l . 1 98 3b ). D e u t e r a t i o n o f t h e a c y l c h a i n s o f D M P C p e r m i t t e d t h eo b s e r v a t i o n o f th e i n t e r a c t i o n o f t h e p r o t e i n w i t h e a c h l ip i d s p e ci es v i a m e a s u r e m e n t o ft h e C - - H a n d C - - Z H s t r e tc h i n g fr e q u en c i es . T h e p e r t u r b i n g e f f ec ts o f t h e p r o t e i n w e r em o r e p r o n o u n c e d o n t h e D P P C c o m p o n e n t t h a n o n t he D M P C - d 5 4 . P r ef er en ti alp a r t it i o n in g o f C a 2 + - A T P a s e i n m i x tu r e s o f p e r d e u t e r a t e d a n d n o n - d e u t e r a t e dp h o s p h o l i p i d s in d if f er e n t p h y s i c a l s t a te s h a s a l so b e e n s h o w n b y F T - I R J a w o r s k y a n dM e n d e l s o h n , 1 9 8 5 ) .

T h i s t y p e o f s t u d y w a s a l so e x t e n d e d t o t h e t e r n a r y s y s te m P S / D P P C - d 6 2 /g l y c o p h o r i n w h e r e i t w a s s h o w n t h a t g l y c o p h o r i n p r e f e r e n ti a l ly i n t e r a c t s w i t h t h e P Sc o m p o n e n t M e n d e l s o h n e t a l . t 9 8 4b ) . F u r t h e r m o r e , a s p e c if ic p e r t u r b a t i o n o f t h ei n t e rf a c ia l re g i o n o f P S w a s d e d u c e d f r o m F o u r i e r d e c o n v o l u t e d s p e c t r a in t h e l i p idC = O s t r e tc h i n g re g i o n . T h e p r e s e n c e o f g l y c o p h o r i n c a u s e d a n i n c re a s e i n th e r e la t i v ei n t e n s i ty o f t h e 1 7 4 2 c m - 1 c o m p o n e n t c o m p a r e d t o t h e p u r e li p id s y s te m a b o v e T m.T h i s s u g g e st s t h a t a h i g h e r p r o p o r t i o n o f t h e a c y l c h a i n s a d o p t a t r a n s c o n f o r m a t i o n

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IR Studies of Me mbranes 245a b o u t C 1 - C 2, I n a d d i t i o n , a n e w c o m p o n e n t w a s o b s e r v e d a t 1 7 1 4 c m - t w h i c hs u g g e st t h a t a p r o p o r t i o n o f t h e s n - 2 c a r b o n y l s a r e i n a m o r e p o l a r e n v i r o n m e n t t h a n i nf r ee P S , p o s s i b l y i n v o l v e d in a w e a k h y d r o g e n b o n d .

O u r s e c o n d - d e r i v a t i v e a n a l y s i s a l s o p e r m i t s a n i n v e s t i g a t i o n o f l i p i d - p r o t e i ni n t e r a c t i o n s a t t h e i n t e r f a c ia l r e g i o n o f t h e b i l a y e r ( L e e et al. 1 98 5b ). T h e s e c o n d -d e r i v a t i v e s p e c t r a r e v e a l t h e sn-1 a n d sn -2 c a r b o n y l s n e a r 1 7 4 0 c m - 1 a n d 1 72 6 c m - i ,r e s p e c ti v e l y . M e a s u r e m e n t o f t h e b a n d i n t e n s i t y r a t i o 11 ~26 I 1 74 0 ( T a b l e i ) i n d i c a t e st h a t t h e i n s e r ti o n o f i n t e g r a l p r o t e i n s i n t o a l i p id b i l a y e r c a u s e s a c o n f o r m a t i o n a lc h a n g e a t t h e in t e rf a c ia l r e g io n b e lo w t h e p h a s e t r a n s it i o n t e m p e r a t u r e ( 23 ~ b u t h a sl it tl e e f fe c t a b o v e t h is t e m p e r a t u r e . T h e p r e s e n c e o f C a 2 + - A T P a s e o r b a c t e r i o r h o d p s i nr e d u c e s t he c o n f o r m a t i o n a l i n e q u i v a l e n c e o f t h e c h a in s s u c h t h a t a p r o p o r t i o n o f t h eo r i g i n a l l y b e n t sn -2 c h a i n s a r e c o n s t r a i n e d i n a c o n f o r m a t i o n s i m i la r t o t h a t o f t h e sn-1c h a i n , l e a d i n g t o a r e d u c t i o n i n b a n d i n t e n s i t y a t 1 7 2 6 c m - 1 ( T a b l e 1). A s ac o n s e q u e n c e t h e p e r t u r b e d sn -2 c a r b o n y l s a r e d i s p la c e d t o w a r d t h e c e n t re o f th eb i l a y e r .

T h e f r e q u e n ci e s o f t h e b a n d s a r is i ng f r o m t h e p h o s p h a t e a n d c h o l in e g r o u p s o fD M P C h a v e b e e n m e a s u r e d i n th e p r e se n c e o f g l y c o p h o ri n ( M e n d e l s o h n et al. 1981).N o s ig n if ic a n t v a r i a t i o n s w i t h t e m p e r a t u r e o r p r o t e i n c o n c e n t r a t i o n w e r e o b s e rv e d .

TaMe 1. The effects of CaZ+-A TPase and bacteriorhodopsin on the intensity ratio of the carbonylstretching bands of DM PC11726/11740

DMPC/Ca 2 -ATPase D MPC/bact eriorhod opsinTemp erat ure/ ~ DM PC 245:1 135:110 0.52 0.31 0.3220 0.45 0.43 - -25 0.50 0.55 --35 0.79 0.70 0.8345 0.80 0.81 --

Da ta we re obtained from the second-derivative pectra presented, n part, by Lee et al. (1985b). The negativeabsorbance intensities at 1726 cm-1 and 1740 cm-1 were measured at eac h temperature.

e m b r a n e P r o t e i n s

P o l y p e p t i d e s a n d C o n f o r m a t i o n a l A s s i g n m e n t sI n f o r m a t i o n o n t h e c o n f o r m a t i o n o f p o l y p e p t i d e s a n d p r o t e i n s m a y b e d e r iv e d

f r o m t h e i r I R s p e c t r a ( K r i m m , 1 9 6 2; Su si , 1 96 9 ; F r a s e r a n d M a c R a e , 1 9 73 ; F a w c e t ta n d L o n g , 1 9 73 ; T h o m a s a n d K y o g o k u , 1 97 7). T h e a s s i g n m e n t o f t h e c h a r a c te r i s ti ca b s o r p t i o n s ( th e s o - c a l l e d a m i d e b a n d s ) h a s b e e n a s s i st e d b y t h e a n a l y s is o f m o d e lc o m p o u n d s s u c h a s N - m e t h y l a c e t a m i d e a n d i s s u m m a r i s e d in t h e se re v ie w s . F o r m a n yy e a r s th e r e w a s a g r e a t d e a l o f c o n t r o v e r s y c o n c e r n i n g t h e m o l e c u l a r d e s c r i p t io n o ft h e s e v i b r a t i o n s a n d t h i s is s u m m a r i s e d b y S u s i ( 19 69 ). I t i s n o w g e n e r a l l y a c c e p t e d t h a tt h e a m i d e I t o a m i d e V I I v i b r a t i o n s c a n n o t b e d e s c r i b e d a d e q u a t e l y b y a n y s i n gl ed i s p l a c e m e n t c o o r d i n a t e . I t s h o u l d b e n o t e d t h a t t h e se a m i d e b a n d s a r e n o t t h e o n l y

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246 Lee and Chap man

absorp t ions obse rved . The IR spec t ra o f po lypep t ides and p ro te ins a l so inc ludecon t r ibu t ions f rom the amino ac id s ide -cha ins .

The am ide I and I I bands a re sensi t ive to the seconda ry s t ruc tu re o f po lype p t idesand p ro te ins and a re the m os t f r equen t ly used m odes in conform at iona l ana lys i s. Thef u n d a me n t a l t h e o r y h a s b e e n d e s c r ib e d b y M i y a z a wa 1 96 0) . S o m e e a rl y a p p l i ca t i o n sof th i s app roac h a re those o f M iyaza wa and B lou t 1961), Kr im m 1962), Sus i e t a l .1967), Tim ashe ff and Susi 1966), and Tim asheff e t a L 1967). These and oth er s tudies ,bo th exper imen ta l and theore t i ca l Jakes and K r imm , 1971 ; Ab e and K r imm , 1972;B a n d e k a r a n d Kr i mm , 1 9 8 0; Kr i mm a n d B a n d e k a r , 1 9 80 ; Dw i v e d i a n d Kr i mm , 1 98 4;Chi rgadze and Nevskaya , 1976a ,b ; Nevskaya and Chi rgadze , 1976 ; Ch i rgadze e t a l .1973 ; Ch i rgadz e and Brazhn ikov , 1974 ; K aw ai and Fa sm an , 1978) have enab ledconf iden t co r re la t ions be tween amide I and I I band f requenc ies and secondaryconformat ion . These a ss ignments a re p resen ted in Tab le 2 .

Table 2. Cha racter is t ic amide I and I I f requencies for var ious polypep tide confo rmatio nsAnide I (cm -1) Am ide I I (cm-1)

C o n f o r m a t io n H 2 0 2 H 2 0e-H elix 1652 (s) 1650 (s) 1546 (s)1646 (w) 1644 (w) 1516 (w)Ran dom coil 1656 1643 1520An ti-para llel 1632 (s) 1632 (s) 1530 (s)Ch ain fl-sheet 1690 (w) 1675 (w) 1510 (w)Parallel ch ain 1630 (s) 1632 (s) 1530 (s)/3-Sheet 1645 (w) 1648 (w) 1550 (w)

s: strong.w: weak.Suspension of soluble proteins in 2H2 0 shif ts the amide I I band (principal ly N -- H bending) to f requenciesnea r 1450cm -~ (amide I I ) .

An e x t en s iv e I R s t u d y o f t h e c o n f o r ma t i o n o f t h e a mp h i p a t h i c p o l y p e p t i d emel i t tin has been m ade Lav ia l le e t a t . 1982). In a qu eo us so lut ion m el i t t in exis ts in amo n o m e r - t e t r a me r e q u i li b ri u m w h i c h is d e p e n d e n t o n b o t h c o n c e n t r a t io n a n d i o n i cs t r e n g t h F a u c o n e t a l . 1979). Ana lys i s o f the am ide I and I I ba nd f requenc ies in H 2 0and 2HzO revea led tha t a t me l i t t in concen t ra t ions in which the t e t r amer ic fo rm i sdom inan t , the confo rma t ion i s ma in ly e -he li ca l a t h igh t em pera tu re 31~ and amix tu re o f f i- and e -he l ica l fo rms a t low tem pera tu re 8~ Lav ia l le e t a l . 1982). Therela t ive in tensi ty of the am ide I I and am ide I band s A~I/A1) wa s high 1 .8-1 .1) for thete t ramer ic fo rm in wa te r com pared to the m ono m er ic fo rm 0 .75). M el i tt in inse r t s in tophosp ho l ip id b i l aye r s in i ts e -he li ca l fo rm Da wso n e t a t . 1978) and in these sam plesam ide I and I I ba nd m ax im a o f 1653 cm - 1 and 1545 cm - x, r e spec tive ly , were ob ta ined .Th is conform at ion was unaf fec ted by the ge l to l iqu id -c rys ta l line phase t r ans i t ion o f thebilayers.The s t ruc tu re o f the po lyp ep t ide an t ib io t ic , g ramic id in A has a l so beeninves t iga ted by IR spe c t roscop y in so lu t ion and a f t e r inse r t ion in to pho spho l ip idbi layers Cort i jo e t a l . 1982; Syche v and Ivano v, 1982; Buchet e t a l . 1985). G ram icidin

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IR Studies of Membranes 247A is a linear polypeptide antibiotic that renders biological membranes and lipidbilayers passively permeable to small monovalent cations by forming a channelstructure (Chappell and Crofts, 1965; Hladky and Hadon, 1970, 1972). It consists offifteen alternating D- and L-amino acids (Sarges and Witkop, 1965). The structure ofthe polypeptide in lipid bilayers is a matter of some dispute and is dependent ontemperature, lipid :protein rat io and lipid class (Sychev and Ivanov, 1982). Although anN-terminal to N-terminal linked dimeric structure has been proposed on the basis ofNMR evidence (Weinstein e t a l . 1979), IR and CD data are consistent with the presenceof an anti-parallel /~-sheet structure in DMPC liposomes at lipid:protein ratiosbetween 350:1 and 35:1 (Sychev and Ivanov, 1982). In DPPC bilayers an anti-paralMdouble-helical structure (Veatch e t a l . 1974) is found at high lipid:protein ratios(Sychev and Ivanov, 1982).

G l o b u l a r P r o t e i n sA question remains concerning the extent to which the conformationalassignments obtained from relatively simple polypeptides and summarised in Table 2can be used to describe the structure of large globular proteins. The first detailed study

of the IR spectra of globular proteins in HaO was performed by Koenig and Tabb(1980) using digital subtraction of the aqueous background. The difference IR spectraof haemoglobin, bovine serum albumin, ribonuclease, fl-lactoglobulin, and e-caesinwere examined in the region 2000-800 cm- 1. The amide I frequency was shown to bethe same in the aqueous and cast film samples. The amide II band was shifted to higherfrequencies on dissolution in water as a result of hydrogen bonding to N--H . Althoughhydrogen bonding had no effect on the amide I maximum there was a shift in thedistribution of the amide I frequencies to lower values which is indicative of increasedhydrogen bonding on dissolution (Susi, 1972). A sharpening of the amide I band ofthose proteins with some e-helical conformation occurred upon dissolution (Koenigand Tabb, 1980). This reduction in bandwidth was interpreted as being due to orderingof e-helical segments in aqueous solution and suggests that e-helical proteins are not intheir native state in the solid phase.

By correlation of the difference spectra with the known secondary structures of theproteins studied, Koenig and Tabb were able to distinguish e-helical, random coil andfl-sheet contributions by a combination of amide I maximum frequency and bandintensity in the amide III region. The amide II peak maxima of-these proteins did notpermit a distinction between the various conformations. However, it was noted that themore ordered proteins (haemoglobin and bovine serum albumin) gave a smaller shiftdue to hydrogen bonding on dissolution than the less ordered proteins (ribonuclease,/lactoglobulin and a-casein).

In principle, a globular protein containing several types of substructure will giveseveral amide I and II maxima. However, the large half-widths of these componentsprevents their resolution. Least squares optimisation and Fourier deconvolutionprocedures have been used for the analysis of overlapping bands (Fraser and Suzuki,1966, 1969; Ruegg e t a l . 1975; Kauppinen e t a l . 1981; Jap e t a l . 1983; Mendelsohn e ta l . 1984a). The usefulness of least squares calculations is limited by the requirement forextensive input information. Typically, the number and peak frequencies of the

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248 Lee and Chapmancomponent bands are required, together with their lineshapes, half-widths, and a linearbaseline. An alternative method for assessing the number and position of thecomponent peaks is derivative spectroscopy (Maddams and Southon, 1982). Second-derivative spectra of water-soluble proteins have recently been obtained (Susi andByler, 1983). These authors were able to resolve peaks associated with a-helical,/%sheet,and fl-turn conformations together with the vibrat ions of some amino acid side-chains.More recently, different denatured forms of water-soluble proteins were resolved usingresolution enhancement and second-derivative calculations (Purcell and Susi, 1984).While other workers use Fourier self-deconvolution as a means of resolutionenhancement (Dev e t a l . , 1984; Yang e t a l . , 1985), our preferred approach is the use ofsecond- (Lee e t a l . , 1985a,b) and fourth-derivatives.Information on the absorption of amino acid side-chains in the 1800-1500 cm-region, i.e., overlapping the amide I and II bands, has been available for some time(Bendit, 1967; Chirgadze e t a l . , 1975). The vibrations of asparagine, glutamine, aspartic,and glutamic acids, arginine and tyrosine may contribute to the spectra of proteins inthis region. With the application of second-derivative methods the resolution of thesebands has become possible (Susi and Byler, 1983; Lee e t a l . , 1985b) thereby providing aprobe into specific regions of globular proteins.

D e t a i l e d S t u d i e s o n B a c t e r i o r h o d o p s i n a n d C a 2 - A T P a s eThe structure of bacteriorhodopsin in the purple membrane of H a l o b a c t e r i u m

h a l o b i u m has been studied by IR spectroscopy (Rothschild and Clark, 1979b; Cortijo e ta l . , 1982; Krimm and Dwivedi, 1982; Lee e t a l . , 1985). These studies have confirmed theexistence of a predominantly a-helical structure for this protein. The observed amide Ifrequency of 1660-1662 cm - ~ s unusually high for e-helices and has been attributed todistorted a-helices (Rothschild and Clark, 1979b) or eii helices (Krimm and Dwivedi,1982; Dwivedi and Krimm, 1984). In the en helix the N- H bonds are tilted inwardtoward the helix axis whereas in the cq structure they are essentially parallel to the axis.Polarised IR spectroscopy has been applied to determine the angle of tilt of the e-helices in dry, oriented specimens (Rothschild and Clark, 1979a). In agreement with theelectron microscope data, the dichroism of the amide A, I and I1 bands indicated anaverage spatial orientation for the helices of less than 26 ~ from the membrane normal.On reconstitution of bacteriorhodopsin in bilayers of D MPC, the major component ofthe amide I, revealed by second-derivative analysis, shifted by 5 cm -1 to lowerfrequency, suggesting a conformational change in the a-helices (Lee e t a l . , 1985). Thisstructure was unaltered by the change in membrane fluidity which is characteristic ofthe phase transition.We present, in Fig. 4, difference (a), second-derivative (b) and fourth-derivative (c)spectra of an aqueous suspension of purple membrane at 20~ after subtraction of theabsorption of the water. The amide I maximum is found at 1660 cm- 1 and may beattributed to distorted or e~-helices. A predominantly e-helical structure is alsoindicated by the amide II maximum at 1545 cm -~, in agreement with the electrondiffraction data of Henderson and Unwin (1975). However, an amide I band shoulder at1630-1640cm -1 suggests the presence of some /~-structure. This absorpt ion isrendered more observable by the second- and fourth-derivative treatments and shownto have a maximum at 1635 cm-1. The appearance of a band at 1684 cm-1 suggests

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IR Studies of Membranes 49

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W VENUMBER cmI)F i g 4 FT -IR spectra of purple mem brane in water at 20~ Spectra wererecorded on a Perkin-Elmer m odel 1750 spectrometerat 2 cm - ~ esolution andapodized with a m ediu m Beer-Norton function. a) Difference spectrumgenerated after subtraction of the spectrum of water, b) second-derivativeof a),c) fourth-derivative of a). Minim a in the second-derivative correspond tomax ima in the difference spectrum which also correspond to maxim a in thefourth-derivative.

t h a t a p r o p o r t i o n o f t h i s e - s t r u c t u r e i s a n t i - p a r a l l e l p l e a t e d s h e e t . B a n d s a t 1 61 8 c m - 1a n d 1 5 1 7 c m - 1 m a y b e t e n t a ti v e l y a s s ig n e d t o t y r o s i n e s i d e -c h a i n a b s o r p t i o n sC h i r g a d z e e t a l . 1 97 5). B a n d s i n t h e r e g i o n 1 4 6 9 - 1 4 3 9 c m - 1 a r e p r i n c i p a l l y d u e t o

C - - H d e f o r m a t i o n m o d e s f r o m t h e li p id a cy l c h a in s .T h e s t r u c t u r e o f t h e s a r c o p l a s m i c r e t i c u lu m S R ) o f r a b b i t s t r i a te d m u s c l e h a s a l sob e e n in v e s t i g a te d b y I R s p e c t r o s c o p y C o r t i jo e t a l . 1 9 82 ; M e n d e l s o h n e t a l . 1 9 8 4 ; L e e

e t a l . 1 9 8 5b ; A r r o n d o e t a l . 1 985 ). E x a m i n a t i o n o f t h e a m i d e I a n d I I m a x i m a i n d ic a t e dt h a t t h e m a j o r p r o t e i n p r e s e n t i n t h i s m e m b r a n e , t h e C a a + - A T P a s e , c o n t a i n su n o r d e r e d a s w e ll a s e -h e l ic a l c o n f o r m a t i o n C o r t i jo e t a l . 1 9 8 2 ; L e e e t a L 1985b) .F o u r i e r d e c o n v o l u t i o n M e n d e l s o h n e t a l . 1 98 4) a n d s e c o n d - d e r i v a t i v e a n a l y s i s L e e e ta l . 1 9 8 5b ) o f t h e a m i d e I r e g i o n re v e a l e d a m i n o r a m o u n t o f / 3 - s h e e t s t r u c t u r e .T e m p e r a t u r e - d e p e n d e n t c h a n g e s i n th e a m i d e I I re g i o n r e v e al e d b y F o u r i e r s e lf -d e c o n v o l u t i o n m a y h a v e b e e n d u e to a r e d u c t i o n i n t h e p r o p o r t i o n o f e - h el i ca ls t r u c t u r e a s t h e t e m p e r a t u r e w a s in c r e a s ed a l t h o u g h t h e in f lu e n c e o f i n c r e as e d 1 H 2 He x c h a n g e w a s n o t e li m i n a t e d M e n d e l s o h n e t a l . 1 98 4). O u r s e c o n d - d e r i v a t i v e L e e e ta l . 1 98 5) a n d f o u r t h - d e r i v a t i v e L e e , D . C . a n d C h a p m a n , D . , u n p u b l i s h e d ) a n a l y s i s o f

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250 Lee and Chapm an

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