and leucine-enkephalin in a membrane environment

THE JOURNAL D 1984 by The American Society of Biological

OF BIOLOGICAL CHEMISTRY Chemists, Inc

Vol. 259. No . 23, Issue of December 10, PP. 14935-14940,1984 Printed in U. S. A.

Evidence for a Folded Conformation of Methionine- and Leucine-Enkephalin in a Membrane Environment*

(Received for publication, May 21,1984)

Basil A. Behnam and Charles M. DeberS From the Research Institute, The Hospital for Sick Children, Toronto, M5G 1x8 and the Department of Biochemistry, University of Toronto, Toronto, M5S 1A8, Ontario, Canada

Transfer of an aqueous-soluble peptide hormone or neurotransmitter such as [Met]- or [Leulenkephalin (Tyrl-Gly2-Gly3-Phe4-Met6(LeuS)), to the lipid-rich environment of its membrane-embedded receptor protein may convert the peptide into a (“bioactive”) conformation required for eliciting biological activity. We have examined by high-resolution nuclear magnetic resonance (NMR) spectroscopy the conformational parameters of free enkephalin in aqueous solution versus those of enkephalin bound to lysophosphatidylcholine micelles using two approaches: 1) exchange rates, line broadening, coupling constants, and chemical shift changes of enkephalin backbone peptide N-H protons were measured for free and membrane-bound peptide in H20 (360 MHz, pH 5.6, 20 “C). A selective upfield shift observed for the Met6(Leu6) N-H proton upon lipid binding was interpreted in terms of its incorporation into an intramolecular H-bond. 2) 13C chemical shift changes induced by the shift reagent praseodymium nitrate (Pr(N03),) were compared in the presence and absence of lipid micelles. Significant changes occurring in Gly’ carbon atoms in membrane-bound enkephalin suggested the relative proximity of this residue to the Pr3+ atom (bound to the Met6(Leu6) COOH-terminal carboxylate 4 residues away). These combined results, in conjunction with studies on the specific interactions of enkephalin substituents with the micelles (Deber, C. M., and Behnam, B. A., (1984) Proc. Nutl. Acad. Sci. U. S. A. 81,61-65) suggest that enkephalin folds into an intramolecularly H-bonded &turn structure (with an H-bond between Gly2 C=O and Met6 NH) in the lipid environment. Such folding could facilitate the positioning of strategic residues in vivo as the hormone diffuses toward its receptor.

Methionine-enkephalin (Tyr-Gly-Gly-Phe-Met) and leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu) are neurotransmit- ters which bind to the same receptor site as rigid opiate agonists (Hughes et al., 1975). Since the secreted neurotrans- mitters encounter both an aqueous phase and a lipid-rich environment en route to their membrane-embedded protein receptors, knowledge of their conformation(s) in each milieu is necessary to our understanding of their function on the molecular level.

* This work was supported, in part, by Medical Research Council of Canada Grant MT-5810 to C. M. D. NMR spectra were recorded at the NMR Centre, which is supported, in part, by Medical Research Council Maintenance Grant MT-6499. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom all correspondence should be addressed.

Efforts to elucidate structure/function relationships in these endogeneous analgesic peptides and their analogs in solution have extensively utilized nuclear magnetic resonance techniques (for a review, see Schiller, 1984). Studies on these pentapeptides in aqueous solution have suggested that the enkephalins exist as an equilibrium ensemble of several un- folded conformers of approximately equal energy, with folded structures occurring only in a relatively low proportion (Bleich et al., 1976, 1977; Fischman et al., 1978 Higashijima et al., 1979; Khaled et al., 1979; Levine et al., 1979; Zetta et al., 1979; Zetta and Cabassi, 1982). In contrast, accumulating evidence from studies in the organic solvent dimethyl sulfoxide has indicated that the zwitterionic form of enkephalin exists in a folded “Type I @-turn” conformation with an intramolecular hydrogen bond between the carbonyl oxygen of Gly’ and the amide proton of the Met’ (or Leu‘) residue (Garbay-Jaure- guiberry et al., 1976, 1977, 1982; Jones et al., 1976; Roques et al., 1976; Stimson et al., 1979; Marion et al., 1981; Zetta and Cabassi, 1982).

Although these data in dimethyl sulfoxide provide a valua- ble adjunct, comparatively few data exist on the conformational behavior of enkephalins specifically in membrane environments where peptide conformational features might be crucial for recognition by receptors. For example, one possible effect lipid might exert in neurotransmitter/receptor events is that of altering the conformational ensemble (uersus that in the aqueous phase) to populate peptide structure(s) which may include a “biologically active” conformation required for receptor binding. Enkephalins have been shown by NMR techniques to bind negatively-charged lipids such as phosphatidylserine via ionic attractions (Jarrell et al., 1980), and, when formal electrostatic attractions are not present, neutral lipids such as lyso-PC’ via hydrophobic interactions (Deber and Behnam, 1984). Enkephalin should be able to bind to membrane surfaces in either linear or folded (&turn) conformations, with the binding favoring peptide structures which place enkephalin hydrophobic substituents on a non-polar face suitable for lipid interactions. Studies to date on lipid- induced changes in enkephalin ‘H and 13C NMR spectral parameters have confirmed such a specificity or “sidedness” for the peptide/lyso-PC and peptide/lysophosphatidylglycerol complexes, as evidenced by differential extents of peptide resonance line broadening and selective chemical shift movements (e.g. in Phe and Tyr side chain @ and aromatic ‘H resonances) (Behnam and Deber, 1983; Deber and Behnam, 1984). However, since peptide NH protons were exchanged in the DzO solvent, no direct information concerning enkephalin backbone conformation(s) was obtained in those studies.

In the present work, the specific effect of the lyso-PC

The abbreviations used are: PC, phosphatidylcholine; ACTH, adrenocorticotropic hormone.

14935

14936 Conformation of Enkephalin in a Membrane Environment

environment on the enkephalin backbone conformation is examined by monitoring spectral behavior of enkephalin amide protons in aqueous (H20) and lipid environments. Furthermore, we have compared the behavior of individual carbon nuclei of the free enkephalin molecule with those of the enkephalin-lipid complex induced by the shift reagent praseodymium nitrate, Pr(N0J3. By combining the data obtained, evidence is presented that enkephalin in a membrane environment likely exists in a folded 2-5 intramolecularly hydrogen-bonded @-turn conformation.

MATERIALS AND METHODS

[Met'IEnkephalin (Tyr-Gly-Gly-Phe-Met) (Bachem), [Leu'lenkephalin (Tyr-Gly-Gly-Phe-Leu) (Fluka), egg L-cu-lysophosphatidylcholine (Sigma), and praseodymium nitrate pentahydrate 99.9% (Alfa), were used without further purification.

'H NMR spectra were determined at 360 MHz using a Nicolet NIC spectrometer operating in the Fourier transform mode with 16K data points and typically 220 accumulations for each spectrum. A 5-s frequency pulse was used to suppress the solvent resonance. Chemical shifts are given in parts/million after standardization of the spectrometer to external tetramethylsilane. 'H NMR measurements were performed on samples with peptide concentration of 8.72 X M (Met-enkephalin = 2.5 mg in 0.5 ml of HzO), prepared in H,O a t 20 "C, pH 5.6. The pH of samples was varied using hydrochloric acid and sodium hydroxide diluted in H,O. pH values were meter readings in Hz0 measured directly in the NMR tubes a t room temperature. Lipid-induced shifts were measured after introducing successive weighed amounts of the lipid into the H20 solution of the peptide.

Proton-decoupled I 3 C NMR spectra were determined at 90 MHz using a Nicolet NIC spectrometer operating in the Fourier transform mode with 16K data points and typically 12,000 accumulations for each spectrum. Chemical shifts are reported in parts/million downfield from internal [2-'3C]acetonitrile as reference standard. Peptide concentrations ranged from 2.73 to 2.82 X lo-' M. Shift reagent experiments in the presence of lipid were performed on samples containing 23.5 mg of Met-enkephalin in 1.5 ml of D,O + 160 mg of lyso-PC; and 23.5 mg of Leu-enkephalin in 1.5 ml of D,O + 118 mg of lyso-PC; 23 "C; pH 6. Lipid concentrations were above critical micelle concentration levels (Kellaway and Saunders, 1970). The pH values are pH meter readings in D,O (Merck, Sharp and Dohme, 99.8%) (uncorrected) measured directly in the NMR tubes at room temperature. For the measurements of Pr(II1)-induced shifts, successive weighed amounts of Pr(N03)3.5H20 were introduced into a solution of the peptide/lipid complex in D,O. For measurements of Pr"+-induced shifts in the free enkephalins, the peptides were dis- solved in D,O (99.8%) solutions a t a concentration of 2.82 X lo-' M in case of Leu-enkephalin and 1.53 X lo-' M in Met-enkephalin experiments to which weighed amounts of P T ( N O ~ ) ~ . ~ H ~ O were introduced.

RESULTS

Proton N M R Spectra of Enkephalin Bound to Phospholipid Micelles-"H NMR spectra (360 MHz) of [Metlenkephalin in H,O (pH 5.6, 20 "C) in the region of the amide protons are shown in Fig. 1. For free Met-enkephalin (Fig.la), assignments of the two doublets to Phe4 (at 8.07 ppm) and Met' (at 7.96 ppm) NH protons, and the triplet at 8.02 ppm to the Gly3-NH proton, were aided by spin-decoupling experiments. These established their connectivity with corresponding C,H protons, and are consistent with the assignments reported for enkephalin NH resonances (Anteunis et al., 1977; Levine et at., 1979; Zetta et al., 1979). Under the conditions of this experiment (Fig. l a ) , the Tyrl-NH; and Gly2-NH protons of free Met-enkephalin were in fast exchange with the solvent (H,O) and were thus not observed. The partial intensities of the Phe4-NH resonance and the Gly3-NH resonances relative to the full intensity of the Met5-NH proton in Fig. l a are likely attributable to transfer of saturation arising from the presaturation (solvent suppression) of the H,O resonance (Campbell et al., 1977). In this context, it is interesting to

Tyrl-NHj Met5 & Gly2

V Phe4 I

(d) 11.9

IC\ 7 1 "'QI IVl 1

PI [ Lipid-OH (b) Lyso-PC I Enk

= 2.9 V

r i ! I

1 ~~ -1 I I

8.4 8.2 8.0 7.8 7j6 PPM FIG. 1. 'H NMR spectrum (360 MHz) of [Met'lenkephalin

(2.5 mg in 0.5 ml of HzO, pH 5.6, 20 "C) in the region of the peptide N-H protons. a, free peptide (concentration = 8.72 X M); b-e, sample a to which successive weighed amounts of the lipid have been introduced (6.0, 15.0, 25.0, and 60.0 mg of egg L-a-lysophosphatidylcholine, respectively). Mole ratios of lipid/peptide are shown in the diagram. Chemical shifts are referenced to external

beled "lipid-OH." tetramethylsilane. The hydroxyl proton resonance of lyso-PC is la-

note that both the rate of exchange and the extent of deshield- ing of Met-enkephalin NH protons increase in the order of the enkephalin sequence from NH2 to COOH terminus (Gly2>Gly3>Phe4>Met5), a result which may be attributable to end group charge effects (at both termini) of the zwitterionic peptide. Corresponding phenomena were observed for the NH protons in identical experiments performed with [Leulenkephalin.

To examine the effects of insertion of the enkephalin into a lipid environment, additions at constant pH of the micelle- forming neutral phospholipid lyso-PC were performed (Fig. 1, b-e). Because of the clarity of NMR spectra obtainable, lipids such as lyso-PC which form relatively lower molecular weight micellar particles (versus vesicular particles) are find- ing increasing use as tools for investigation of peptide- and protein/membrane interactions (Brown, 1979; Hagen et al., 1979; Hughes et al., 1982). During the lipid titration, the Met- enkephalin Gly2-NH resonance, which was unobservable in the absence of lipid (Fig. la) , appeared gradually with increasing lipid concentration. The unphased signal which appears near 8.35 ppm in Fig. 1, d and e, is attributable to the Tyrl- NH: protons, folded into this spectral region from a low field position and superimposed over the Gly' signal. The regaining of the full amide proton intensities as a function of succes- sively increasing amounts of lyso-PC is indicative of the reduced exchange rate (with H20) arising upon transfer of the corresponding enkephalin residues into the less polar lipid environment. The resonance line broadening observed in Fig.

Conformation of Enkephalin in a Membrane Environment 14937

7.6 I I I

0 10 20 30 40 MOLE RATIO, LYSO-PC lENK

FIG. 2. Chemical shifts of [Metlenkephalin (M and [Leu] enkephalin (L) amide proton resonances as a function of added lysophosphatidylcholine. Data were obtained from 360 MHz 'H NMR spectra under the conditions given for Met-enkephalin as described in the legend to Fig. 1 (H20, pH 5.6, 20 " C ) .

OH :::a +32

-2ec, 11

+N"3,C,"N \c-15

I +49c=o

o,.+=, 0- C I

N I I

c - 2 4

/ -12

-26 c 11 I 0 c - "

- 1 3 0 - 6

+n ' + 32

FIG. 3. 13C chemical shift changes (Hz) displayed by [Leu] enkephalin upon addition of lysophosphatidylcholine micelles (5-fold molar excess). Spectra were recorded at 90 MHz, pH 6, 23 "C; see "Materials and Methods" for further details. No conformational implications are intended in this diagram.

1 on moving from spectrum a to e provides further confirma- tion of the binding of the aqueous-soluble enkephalin to the lyso-PC micellar particle.

Differential chemical shift changes, both in extent and direction, occur in Met-enkephalin and Leu-enkephalin (spectra not shown) peptide NHs upon addition of lipid, as presented graphically in Fig. 2. While the Phe4-NH and Gly3- NH shifts in both Met-enkephalin and Leu-enkephalin are qualitatively parallel at a mole ratio of 28.6:l lyso-PC/enkephalin (for Phe4-NH, A6 = +50 and +59 Hz, respectively; for Gly3NH, A6 = -25 and -25 Hz, respectively), the Leu- enkephalin Leu5-NH shift of +lo8 Hz is significantly greater than the Met-enkephalin Met5-NH shift of +76 Hz. We attribute the latter result to an enhanced affinity of the hydrophobic leucine side chain for the membrane uersus the sulfur-containing methionine side chain. The incrementally greater shifts observed for Met5-NH and Leu5-NH protons uersus other NH protons are discussed in further detail below.

Effect of the Shift Reagent Praseodymium Nitrate on 13C

Spectra of EnkephalinlLipid C~rnplexes-~~C chemical shift changes induced in Leu-enkephalin resonances in the pres-

ence of 5-fold molar excess of lyso-PC are summarized in Fig. 3. The selective chemical shift changes in Leu-enkephalin resonances reflect a combination of the conformational and environmental changes experienced by the peptide upon association with lipid. The stepwise movements of enkephalin 13C (and 'H resonances, e.g. Fig. 1) during lipid titrations indicate that the system is in fast exchange on the NMR time scale.

We have carried out a series of I3C NMR experiments in which chemical shift movements were compared of individual carbon nuclei of free Leu-enkephalin and of membrane-bound Leu-enkephalin in the presence of the shift reagent praseodymium nitrate (Pr(N03)3) (Fig. 4). For axially symmetric complexes, the pseudocontact shift is proportional to (3 cos2 0, - l)/r? where Oi is the angle between the principal magnetic axis and the distance vector, r,, joining the particular nuclei, in the complexed substrate to the metal (for a review, see Cockerill et al., 1973). Addition of Pr(N03)3 to an aqueous solution of free Leu-enkephalin produced the chemical shift changes shown as the upper set of numbers in Fig. 4. Atoms adjacent to the COOH-terminal Leu residue displayed the greatest shifts (e.g. 881 Hz in Leu CJ, confirming that the praseodymium atom forms a chelate complex with Leu-enkephalin through its coordination to the Leu carboxylate acid group; the negatively charged carboxylate acid group is ex- pected to be a more effective coordination site than the carbonyl site of amide groups (Levine and Williams, 1975; Levine et al., 1979). The observed induced shifts are downfield in all carbon nuclei with the exception of Gly2 C, which was shifted slightly upfield.

Parallel behavior was observed in similar experiments with [Metlenkephalin. At a Pr3+/Met-enkephalin ratio of 1:1, chemical shift changes in observable Met-enkephalin carbonyl carbon resonances were Phe C=O, -72 Hz; Gly3 C=O, -19 Hz; Gly2 C=O, -10 Hz; and Tyr C=O, -5 Hz, while the Met C, resonance moved 544 Hz downfield. At a Pr3+/Met- enkephalin ratio of about 21, corresponding values were qualitatively comparable to the values shown in Fig. 4 for Leu-enkephalin resonances.

Addition of P T ( N O ~ ) ~ to Leu-enkephalin in the presence of

on

0 - 0- \\ -/ C

N 1-15

C+lB

,"

+12 - 4 +11

FIG. 4. "C chemical shift changes (Hz) of free and micelle- bound [Leulenkephalin induced by addition of the shift reagent praseodymium nitrate (Pr(N03)3). Spectra were recorded at 90 MHz, pH 6, 23 "C; see "Materials and Methods" for further details. Mole ratio of Le~-enkephalin/Pr(NO~)~ = 1:l. The upper numbers at each carbon atom are those obtained for free Leu-enkephalin (23.5 mg/1.5 ml in DzO); the lower numbers are those obtained in the presence of lysophosphatidylcholine (118 mg). Negative values are downfield shifts. No conformational implications are intended in this diagram.


5-fold molar excess lyso-PC gave the chemical shift changes shown as the lower set of numbers in Fig. 4.' I t should be noted that the Leu-enkephalin/lyso-PC complex is unlikely to be axially symmetric; since a sign change occurs for values of 0 between 54.7" and 125.3" (Cockerill et al., 1973), the directions of the induced shifts will have an angular dependence. Indeed, upfield shifts are observed for several carbons of membrane-bound Leu-enkephalin. Furthermore, Pr3+ ions are likely to bind to the negatively charged oxygen atoms of lyso-PC phosphodiester head groups as well as to Leu-enkephalin carboxylate sites. For example, Pr3+-induced shifts observed (in the same experiment as presented in Fig. 4) for lyso-PC carbons four bonds distant in either direction from the phosphodiester binding site were 102 Hz (choline N-CH, head group carbon) and 20 Hz (glycerol backbone methine carbon). These values are of the same order of magnitude as the four-bond Pr3+-induced shifts observed in Leu-enkephalin resonances (94 Hz, Leu y-carbon; 35 Hz, Phe carbonyl carbon) (Fig. 4). Despite the binding of Pr3+ ions to sites on lipid as well as peptide, both the r-3 dependence of Pr3+-induced shifts and the rapid equilibration on the NMR time scale among species ensure that shifts observed in Leu-enkephalin resonances reflect direct Pr3+-peptide carboxylate interactions. Interpretation of Pr3+-induced shifts in the presence of lipid is presented below.

DISCUSSION

Endogeneous phospholipids in uiuo, such as those in the microenvironment of the enkephalin receptor, could conceiv- ably play any of several roles in mediating the transfer of a neurotransmitter or peptide hormone from the aqueous to the membrane phase. These could include: facilitating the cap- ture, entry, and concentration of the aqueous-soluble hormone or neurotransmitter into the lipid-rich environment of the receptor; and/or orienting the peptide in the membrane vis u vis the receptor by restricting molecular motions (Deber and Behnam, 1984). In a more specific function, the decrease in local polarity during transfer may promote the conversion of the neurotransmitter into a population of folded, intramolecularly hydrogen-bonded conformation(s), a circumstance which could well be required for eliciting biological activity. The present exploration of the relationship between (aqueous) solution conformation and lipid-bound conformation of enkephalin has provided evidence for such folding.

Conformation of Enkephalin in the Lipid Enuironment- Variations are observed both in extent and direction of amide chemical shifts suggesting a specific combination of not only environmental but also conformational consequences result- ing from interaction(s) of enkephalin with the lipid matrix. In aqueous solution, all NH protons of free peptide in prin- ciple are initially solvated (H-bonded) to water molecules. The lipid-induced upfield shifts such as those which arise in Phe and Met NH resonances (Fig. 1) are thus attributable to the decreased average polarity of the lipid/water environ-

Some resonances of Leu-enkephalin (for example, Phe and Gly2 carbonyl carbons) appear to return to positions near those of free Leu-enkephalin upon addition of Pr3' (compare Figs. 3 and 41, raising the possibility that the increase in the ionic strength of the medium may displace (some) peptide from the lipid. In this connection, Jarrell et al. (1980) have shown that under conditions of maximal interaction (pH 4.0) of enkephalin with (negatively-charged) phosphatidylserine, the addition of 1 M sodium chloride disrupted the largely electrostatic peptide/phosphatidylserine interaction and thus caused the spectrum to revert to that of free enkephalin. However, since the interactions stabilizing the enkephalin/lyso-PC complex are largely hydrophobic, and the concentration of Pr3+ ions is (0.03 M (Fig. 4), displacement of peptide from lipid by Pr(NO& is unlikely to be a significant factor.

ment (uersus pure water) and concomitant decrease in water solvation of peptide NHs. However, since an intramolecular H bond is characteristically less linear and has greater C=O . . . . H-N separation than the stronger H-bond to water, its NH resonance could experience an additional shift to higher field. Thus, the incremental chemical shift to higher field observed for the Met5-NH (86 Hz) uersus the Phe NH (55 Hz) a t 38:l lyso-PC/Met-enkephalin (Fig. 2) is consistent with the incorporation of the Met-NH proton into an intramolecular H bond (to the Gly' carbonyl group) to produce a Gly2-Gly3-Phe4-Met5 P-turn. Concomitantly, the fact that the Gly3-NH resonance experiences a downfield shift (26 Hz) is similarly indicative of a conformational transition. Further support for this suggestion is obtained from Fig. 3, where it may be noted that the largest lipid-induced chemical shift changes (particularly in Gly' C=O) occur in regions of Leu- enkephalin which are involved in the proposed folding of the molecule.

'J(H"-HO) side chain vicinal coupling constants in Met- enkephalin during lipid titration had been found to be invariant for resonances not obscured by lipid (Phe, Met, and Tyr @-proton resonances; Met y-proton resonances; and the Phe a-proton resonance) (Behnam and Deber, 1983). Similarly, no significant variations in the 3J(H"-NH) values of Met5, Phe4, and Gly3 coupling constants were observed upon binding enkephalin to lyso-PC (Fig. 1, b-d). For free Met-enkephalin in H20, 3J(Ha-NH) values, as measured from Fig. la, were Met', 7.6 Hz; Phe4, 7.4 Hz; and Gly3, Z = 12.2 Hz. (For the Gly3 residue, the sum of the vicinal coupling constants is given, i.e. 3J(H"-NH) = 3J(H"~-NH) + 3J(H"2-NH).) Corre- sponding values for free Leu-enkephalin in H,O (spectra not shown) were Leu5, 7.8 Hz; and Phe4, 8.0 Hz. These 3J(H"- NH) coupling constants remained invariant 50.2 Hz up to about 20-fold excess lyso-PC, after which resonance line broadening precluded their accurate determination. The observed values of 3J(H"-NH) are nevertheless typical for P- turn J values (Gierasch et al., 1981), as folding of the peptide backbone need not, on the average, be accompanied by major redistribution of low-energy side chain rotamer states. To the extent that enkephalin spectral parameters in dimethyl sulfoxide solutions can be compared to those in the present lipid/ water mixture, we have compiled literature data from other studies in which a folded P-turn conformation has been proposed for enkephalins in dimethyl sulfoxide (Garbay-Jaure- guiberry et al., 1976; Jones et al., 1976; Stimson et ai., 1979) (Table I). Note that Gly3 and Phe4 chemical shifts crossover as a result of the influence of the lipid environment on the hydrophobic Phe side chain.

Experiments with Pr(N03)3 produced the upper set of numbers on Leu-enkephalin carbons in Fig. 4. Pr3+-induced 13C chemical shift changes for individual carbon atoms in Leu- enkephalin were observed to fall off monotonically from the peptide COOH to NH, terminus. These chemical shift changes, which are presented graphically in Fig. 5 along the Leu-enkephalin carbon backbone, provide further experimen- tal evidence that free Leu-enkephalin exists on the average in aqueous solution in an extended linear conformation (or an ensemble of closely related structures).

Thus, Pr3+-induced shifts should diminish with increasing distance from the Leu-enkephalin COOH terminus unless specific conformational changes in the peptide have altered r values. That this has indeed occurred can be seen clearly in Fig. 5, where Leu-enkephalin carbon atoms 4 residues distant from the COOH terminus (e.g. Gly' C,, and C=O) display a significant increase in Pr3+-induced shifts versus free peptide. These results indicate an enhanced proximity to Pr3+ of the

Conformation of Enkephalin in a Membrane Environment 14939

TABLE I ' H N M R parameters for /Met]- and Ileulenkephalin peptide NH

protons in folded conformations All chemical shifts versus tetramethylsilane, kO.05 ppm. M = Met;

I, = Leu.

Solvent

Chemical shifts 3J(HP-NH) cou- vlina constants . .

ppm HZ Dimethyl sulfoxide-d" 8.0 8.2 7.9 (M) 8.6 7.4 (M) Dimethyl sulfoxide-&* 7.9 8.2 7.8 (M) 8.3 7.2 (M) Dimethyl sulfoxide-@ 8.0 8.2 8.0 (L) 8.4 7.9 (L) Lvso-PC/H2Od 8.1 7.9 7.8 (M) 7.2 7.4 (M) I,yso-PC/HZO' 8.1 7.9 7.6 (L) 7.4 7.1 (L)

Garhay-Jaureguiherry et al. (1376) (300 MHz). h.Jones et al. (1976) (270 MHz). "Stimson et 01. (1979) (2SO MHz). dTh i s work (360 MHz). Chemical shifts at 28.6:l Ivso-PC/Met-

"This work. Chemical shifts at 28.6:l Ivso-PC/Leu-enkephalin; enkephalin (Fig. le): coupling constants at 11.9:l (Fig. Id).

coupling constants at 1191.

I 2

- N

0 .'.i 100

i

i Free peptide

I

C' Ca C' ca C' Ca C' ca C' Leu --he -GGy3-Gly2-Tyr

ENKEPHALIN BACKBONE CARBONS FIG. 5. Praseodymium-induced I3C chemical shift changes

(absolute values, Hz) in Leu-enkephalin backbone carbon atoms. Data were obtained from samples prepared as described in the legend to Fig. 4.

NH2-terminal portion of the enkephalin molecule in the lipid- rich environment versus its corresponding location in aqueous solution. Also, the affected Leu-enkephalin carbons are pre- cisely those which would approach the COOH-terminal end of the molecule in a folded (&turn) structure.

[Metlenkephalin carbons behaved similarly in contact with

Pr3+ in a lipid environment. In one ' T experiment, performed a t 20 MHz, with a mole ratio of Pr"'/Met-enkephalin = 1:3, changes in Met-enkephalin carbonyl carbons (expressed in Hz at 90 MHz) were: Phe C=O, -48 Hz; Gly:' C=O, -20 Hz; Gly' C=O, -48 Hz; and Tyr C=O, -5 Hz.

Orientation of Enkephalin in the Lipid Micelle-Results from the combined investigations presented herein lead to the proposal of a Type I (2-5) @-turn average conformation (for @-turn nomenclature, see Venkatachalam (1968)) for the peptide upon its binding and incorporation into a lyso-PC micelle. A photograph of a Corey-Pauling-Koltun molecular model of such a structure is presented in Fig. 6, where the Leu5 N-H (or analogously the Met5 N-H) of enkephalin participates in an intramolecular hydrogen bond to the Gly2 peptide carbonyl group. Based upon additional information already available from specific chemical shift and line width dependencies of enkephalin 'H and "C resonances (Deber and Behnam, 1984), portions of the side chain substituents of Tyr, Phe, and Leu are deduced to be embedded in the lipid matrix (indicated schematically by the overlay of lyso-PC molecules) while the Leu (Met) carboxylate is just below the head groups of the lipid in a position corresponding to a lyso-PC phosphate group. The zwitterionic peptide is thus specifically bound into the membrane with its negative end facing water while inter- vening hydrophobic substituents sag inward towards the lipid hydrophobic interior. Viewed in the context of a biological membrane, where the positively-charged peptide NH, terminus can also bind electrostatically to phosphate groups of the anionic lipids present, these results reinforce the "attraction- interaction" model we have proposed for hormone/membrane association (Deber and Behnam, 1984), and demonstrate that information contained in the sequence of an amphiphilic peptide can produce binding and induction of secondary structure in an encounter with a complementary amphiphile, the membrane. In these respects, our findings are conceptually analogous to the "address-message'' model, determined by photolabeling and infrared attenuated total reflection spectroscopy techniques, for binding of the peptide hormone ACTHI.24 to neutral and anionic vesicles (Gremlich et al.,

FIG. 6. Photograph of a Corey-Pauling-Koltun molecular model of aconformation of [Leulenkephalin containing a Type I (2-5) @-turn involving GlyZ-Gly3-Phe4-Leu5 residues. The Leus N-H is intramolecularly H-bonded to the Gly2 carbonyl group. A portion of the lysophosphatidylcholine micelle is shown schematically as an overlay to indicate the orientation and degree of penetra- tion of the peptide into the membrane environment as suggested by NMR data (see text). While the model contains bond angles and side chain rotamer positions consistent with this experiment, this static presentation of a dynamic situation should be regarded only as typical of this category of conformations.


1984; Gysin and Schwyzer, 1984). Although the enkephalin/ lyso-PC system cannot mimic the complexity of the situation encountered by functioning enkephalin molecules in vivo, and consists of lipids in monolayer rather than bilayer structures, the conformation deduced for the micelle-bound neurotransmitter is clearly one which provides for its facile entry and specific orientation into a membrane phase.

Acknowledgment-We thank Alan Lee for technical assistance.

REFERENCES Anteunis, M., Lala, A. K., Garbay-Jaureguiberry, C., and Roques, B.

P. (1977) Biochemistry 16, 1462-1466 Behnam, B. A,, and Deber, C. M. (1983) in Peptides: Structure and

Function, (Hruby, V. J., and Rich, D., eds) pp. 445-450, Pierce Chemical Co., Rockford, IL

Bleich, H. E., Cutnell, J. D., Day, A. R., Freer, R. J., Glasel, J. A., and McKelvy, J. F. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2589- 2593

Bleich, H. E., Day, A. R., Freer, R. J., and Glasel, J. A. (1977) Biochem. Biophys. Res. Commun. 74,592-598

Brown, L. R. (1979) Biochim. Biophys. Acta 557, 135-148 Campbell, I. D., Dobson, C. M., and Ratcliffe, R. G. (1977) J. Magn.

Cockerill, A. F., Davies, G. L. O., Harden, R. C., and Rackham, D.

Deber, C. M., and Behnam, B. A. (1984) Proc. Natl. Acad. Sci. U. S.

Fischman, A. J., Riemen, M. W., and Cowburn, D. (1978) FEBS Lett. 94,236-240

Garbay-Jaureguiberry, C., Roques, B. P., Oberlin, R., Anteunis, M., and Lala, A. K. (1976) Biochem. Biophys. Res. Commun. 71, 558- 565

Garbay-Jaureguiberry, C., Roques, B. P., Oberlin, R., Anteunis, M., Combrisson, S., and Lallemand, J. Y. (1977) FEBS Lett. 76, 93- 98

Garbay-Jaureguiberry, C., Marion, D., Fellion, E., and Roques, B. P.

Res. 27,455-463

M. (1973) Chem. Reu. 73,553-588

A. 81, 61-65

(1982) Intl. J . Pept. Protein Res. 20, 443-450

E. R. (1981) Biochemistry 20, 4730-4738

istry 23,1808-1810

Gierasch, L. M., Deber, C. M., Madison, V., Niu, C.-H., and Blout,

Gremlich, H.-U., Fringeli, U.-P., and Schwyzer, R. (1984) Biochem-

Gysin, B., and Schwyzer, R. (1984) Biochemistry 23, 1811-1818 Hagen, D. S., Weiner, J. H., and Sykes, B. D. (1979) Biochemistry

18,2007-2012 Higashijima, T., Kobayashi, J., Nagai, U., and Miyazawa, T. (1979)

Eur. J . Biochem. 97,43-57 Hughes, J., Smith, T. W., Kosterlitz, H. W., Forthergill, L. A,,

Morgan, B. A., and Morris, R. H. (1975) Nature (Lond.) 258,577- 579

Hughes, D. W . , Stollery, J . G., Moscarello, M. A., and Deber, C. M. (1982) J. Biol. Chem. 257,4698-4700

Jarrell, H. C., Deslauriers, R., McGregor, W. H., and Smith, I. C. P. (1980) Biochemistry 19, 385-390

Jones, C. R., Gibbons, V. A., and Garsky, V. (1976) Nature (Lond.) 262,779-782

Kellaway, I. W . , and Saunders, L. (1970) Chem. Phys. Lipids 4, 261-

Khaled, M. A., Urry, D. W., and Bradley, R. J. (1979) J. Chem. SOC.

Levine, B. A,, and Williams, R. J. P. (1975) Proc. R. SOC. Lond. A345,

Levine, B. A., Rabenstein, D. L., Smyth, D., and Williams, R. J. P.

Marion, D., Garbay-Jaureguiberry, C., and Roques, B. P. (1981)

Roques, B. P., Garbay-Jaureguiberry, C., Oberlin, R., Anteunis, M.,

Schiller, P. W . (1984) in The Peptides (Udenfriend, S., and Meien-

Stimson, E. R., Meinwald, Y. C., and Scheraga, H. A. (1979) Biochem-

Venkatachalam, C. M. (1968) Biopolyers 6, 1425-1436 Zetta, L., Cabassi, F., Tomatis, R., and Guarneri, M. (1979) Eur. J .

Zetta, L., and Cabassi, F. (1982) Eur. J . Biochem. 122,215-222

268

Perkin Trans. 11, 1693-1699

5-22

(1979) Biochim. Biophys. Acta 579, 279-290

Biochem. Biophys. Res. Commun. 101, 711-718

and Lala, A. K. (1976) Nature (Lond.) 262, 778-779

hofer, J., eds) Vol. 7, Academic Press, New York, in press

isfry 18,1661-1671

Biochem. 95,367-376

and leucine-enkephalin in a membrane environment

Documents