Proc. Nati. Acad. Sci. USAVol. 81, pp. 61-65, January 1984Biochemistry

Role of membrane lipids in peptide hormone function: Binding ofenkephalins to micelles

(NMR/peptide-lipid interactions/opioid peptides/attraction-interaction model)

CHARLES M. DEBER*t AND BASIL A. BEHNAM**Research Institute, The Hospital for Sick Children, Toronto, ON, M5G 1X8, Canada; and tDepartment of Biochemistry, University of Toronto, Toronto, ON,M5S 1A8, Canada

Communicated by Elkan R. Blout, September 9, 1983

ABSTRACT In the course of their biological function,peptide hormones must be transferred from an aqueous phaseto the lipid-rich environment of their membrane-bound recep-tor proteins. We have investigated the possible influence ofphospholipids in this process, using 360-MHz 'H and 90-MHz13C NMR spectroscopy to examine the association of the opioidpeptides [Met]- and [Leu]enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) with phospholipid micelles. Binding of peptides to lipidwas monitored in NMR spectra by selective chemical shiftmovements (e.g., the Phe aromatic ring protons) and residue-specific line broadening (e.g., of Met/Leu carbonyl- and a-carbon resonances). Results established that the zwitterionichormones associate hydrophobically both with a neutral lipid(lysophosphatidylcholine) and (also electrostatically) with anegative lipid (lysophosphatidylglycerol). An association coh-stant of Ka = 3.7 x 101 M-1 was calculated for the hydropho-bic binding of enkephalin to lysophosphatidylcholine. NMRdata suggested that enkephalin binds to the lipid with Met/Led, Phe, and likely Tyr side-chain substituents associatedwith nonpolar interior regions of the micelle, whereas theCOOH-terminal carboxylate moiety of the peptide is located inthe surface of the lipid particle. An "attraction-interaction"model is proposed for hormone-lipid association wherein neg-ative lipids attract the hormone electrostatically, while site-specific hydrophobic contacts facilitate its entry, concentra-tion, and orientation into the lipid phase.

Aqueous-soluble proteins and peptide hormones are oftenbound to or transferred into membranes in conjunction withtheir biological functions. Because a hormone as well as itsmembrane-embedded receptor protein contain potentialsites of association with lipids, one may hypothesize thatsurrounding endogenous phospholipids could potentiallyplay any of several roles in mediating the transfer. Thesecould include: facilitating the capture, entry, and concentra-tion of the aqueous-soluble hormone or neurotransmitterinto the microenvironment of the receptor; orienting the pep-tide in the membrane vis-d-vis the receptor by restricting mo-lecular motions; and/or, in a more specific function, con-verting the hormone into a conformation required for elicit-ing biological activity.These circumstances are relevant to the enkephalins-the

peptide neurotransmitters that compete with morphine andits derivatives for opiate binding sites in the brain (1). Toexamine the above possibilities, we have initiated a study ofsome enkephalin-lipid complexes to determine (i) whetherhydrophobic interactions as well as electrostatic attractionscontribute significantly to their stabilization, particularly incomplexes with zwitterionic (net neutral) lipids, and (ii) theextent to which these interactions influence hormone mo-tional and conformational parameters.

Physicochemical studies of the association of materialssuch as insulin (2), glucagon (3), and the apolipoproteins (4)with membrane preparations in vitro have been performed todetermine the specific manner through which phospholipidscontribute to their functioning in vivo. Enkephalin itself hasbeen shown to associate through ionic attraction to negativelipids such as phosphatidylserine (5). Using high-resolution1H and 13C NMR techniques, we have now obtained evi-dence for hydrophobic interaction of two endogenous

1 2 3opioid peptides, [Met]- and [Leu]enkephalin (Tyr-Gly-Gly-4 5

Phe-Met/Leu), with a neutral phospholipid, lysophosphati-dylcholine (lysoPtdCho). The peptides also interact througha combination of electrostatic and hydrophobic interactionswith the anionic lipid lysophosphatidylglycerol (lysoPtd-Gro). In addition, we have been able to infer specific sites onthe peptide of major interaction with lipid and to obtain aquantitative measure of the hydrophobic component of pep-tide-lipid interaction. The possible role(s) of endogenousphospholipids is evaluated on the basis of these data.

MATERIALS AND METHODS[Met]Enkephalin (Tyr-Gly-Gly-Phe-Met; Bachem FineChemicals, Torrance, CA), [Leulenkephalin (Tyr-Gly-Gly-Phe-Leu; Fluka, Hauppauge, NY), egg L-a-lysoPtdCho (Sig-ma), L-a-lysoPtd-DL-Gro (Sigma), and praseodymium nitratepentahydrate (99.9%; Alfa-Ventron, Danvers, MA) wereused without further purification.NMR samples were prepared with a peptide concentration

of 8.72 mM for 1H NMR and from 27.3 to 28.2 mM for 13CNMR studies. Lipid concentrations, given in figure legends,were generally above critical micelle concentration levels(6). The quoted pH values are pH meter readings in 2H20(Merck Sharp and Dohme, Montreal; 99.8%) that were un-corrected for the deuterium isotope effect and were mea-sured directly in the NMR tubes at room temperature.1H NMR spectra were determined at 360 MHz with the

Nicolet NIC spectrometer operating in the Fourier transformmode with 16,000 data points and typically 250 accumula-tions for each spectrum. A 5-sec frequency pulse was used tosuppress the residual 1H2HO resonance. Temperature was23 ± 10C. Chemical shifts are given in ppm after standardiza-tion of the spectrometer to external tetramethylsilane. Lipid-induced shifts were measured after introducing successive,weighed amounts of the lipid into the 2H20 solution of pep-tide.

'H-decoupled 13C NMR spectra were determined at 90MHz with the Nicolet NIC spectrometer operating in theFourier transform mode with 16,000 data points and typical-ly 12,000 accumulations for each spectrum. Chemical shiftsare reported in ppm downfield from internal [2-'3C]acetoni-trile reference standard.

Abbreviations: PtdCho, phosphatidylcholine; lysoPtdCho, lyso-phosphatidylcholine; lysoPtdGro, lysophosphatidylglycerol.

61

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62 Biochemistry: Deber and Behnam

For the measurements of Pr(III)-induced shifts, the pep-tides were dissolved in 2H20 at 28.2 mM in the case of[Leu]enkephalin and 15.3 mM in the [Met]enkephalin experi-ment. Successive weighed amounts of Pr(NO3)3 5H2O wereintroduced into a solution of the peptide in 2H20.

RESULTS13C NMR Spectra of Enkephalins and Phospholipids. 13C

NMR spectra (90 MHz) of [Met]enkephalin carbonyl car-bons in the presence of increasing concentrations of the neu-tral (zwitterionic) lipid lysoPtdCho are shown in Fig. LA Be-cause of the clarity of spectra obtainable, this lipid and oth-ers that form micellar particles are emerging as valuabletools for investigation of protein-membrane interactions (9-12). Significant upfield chemical shift changes (A8) were ob-served for three of the five enkephalin carbonyl carbons(Gly-2, Phe, and Met), while the NH2-terminal Tyr and Gly-3carbonyl-carbon resonances displayed little or no change upto approximately 5-fold molar excess of lipid (Fig. 1, spec-trum D).t

Binding of [Leu]enkephalin to lysoPtdCho micelles pro-duced similar results (Fig. 2B), whereas the chemical shiftchanges observed for the titration of [Leu]enkephalin withmicelles prepared from the negatively charged lipidlysoPtdGro are shown in Fig. 2A. In the latter experiment,the chemical shift of the NH2-terminal Tyr carbonyl-carbonresonance shifted downfield from 169.1 to 169.9 ppm afteraddition of the equivalent amount of lysoPtdGro. This be-havior should be contrasted with the negligible chemicalshift change for Tyr carbonyl-carbon resonances of both[Met]- and [Leu]enkephalin over the range of 0 to 6-fold mo-lar excess of lysoPtdCho (Fig. 1, spectrum D and Fig. 2B). Itis emphasized that throughout all additions of lipids, the pHremained constant ±0.1 unit. The Gly-3 carbonyl-carbonresonance also reflected the "titration" behavior between 0and 1:1 molar ratio of lysoPtdGro/peptide (Fig. 2A) vs. itsrelative insensitivity to added lysoPtdCho (Fig. 2B). Anoverall comparison of the trends above 1:1 lysoPtdGro/peptide in Fig. 2A with Fig. 2B suggests that the upfieldmovements of Leu, Phe, and Gly-2 carbonyl-carbon reso-nances are comparable, indicating that the enkephalin-lipidinteractions manifested by these shifts are taking place in acorresponding manner with both lipids, being masked, in ef-fect, by the initial primary attraction apparent in the lysoPtd-Gro system.The chemical shift changes upon binding lysoPtdCho mi-

celles are accompanied by general line broadening (Fig. 1,spectra A-D), but additional selective line broadening clear-ly occurs for the COOH-terminal Met carboxylate-carbonresonances [linewidth (uncorrected) = 3.3 Hz (Fig. 1, spec-trum A) for the free [Met]enkephalin and 9.1 Hz for boundpeptide (Fig. 1, spectrum D)]. Parallel effects are observedfor both [Met]- and [Leu]enkephalin with both lysoPtdCho

tThe two resonances designated in Fig. 1 as Gly-2 and Gly-3 corre-spond to assignments inferred (7) from a series of 13C NMR spectraof enkephalins and several analog peptides. We have obtained addi-tional support for these assignments from a study of effects on 13CNMR spectra of [Met]- and [Leu]enkephalin in aqueous solutionupon binding to paramagnetic praseodymium ions. In the presenceof ca. 5-fold excess Pr(NO3)3, the resonance labeled Gly-3 moved0.56 ppm downfield, vs. a change of 0.33 ppm for that labeled Gly-2, as expected for decreasing proximity to Pr3+ bound at the[Met]enkephalin COOH-terminal carboxylate group. Paralleltrends were observed for the pair of Gly a-carbon resonances; theupfield resonance shifted 0.22 ppm, while the downfield resonancewas unaffected. This behavior is in accord with expectations fromthe absolute assignments of these resonances to Gly-3 and Gly-2 a-carbons, respectively, obtained from the NMR spectra of 13C-en-riched synthetic [Met]enkephalin samples (5) (see also ref. 8).Spectra of [Leu]enkephalin and Pr3+ showed qualitatively parallelshifts.

B. +35 mg

lysoPtdCho

Wetl'dE <

APhe

177 175 173 171 169PPm

FIG. 1. Regions of carbonyl-carbon resonances in 13C NMRspectra (90 MHz) of [Met]enkephalin (23.5 mg in 1.5 ml of 2H20, pH- 6) (spectrum A) to which increasing portions of lysoPtdCho havebeen added (spectra B-D). Mole ratios of lipid/peptide range fromca. 2 in spectrum B to ca. 6 in spectrum D. Resonances offree pep-tide (spectrum A) were assigned in accordance with Khaled et al.(ref. 7; see also footnote t). Numbers in spectrum D indicate thetotal chemical shift changes (Hz) from spectrum A to spectrum D;positive shifts are upfield. _L, ester carbonyl-carbon resonance oflysoPtdCho.

and lysoPtdGro. Similar phenomena are observed in the a-carbon region of micelle-bound peptides (Fig. 3); for exam-ple, the [Leu]enkephalin a-carbon resonance is selectivelybroadened in the presence of lysoPtdGro [linewidth = 3.5Hz for free [Leu]enkephalin (Fig. 3, spectrum A) and 10.5Hz at 1:1 lysoPtdGro/peptide (Fig. 3, spectrum B)].'H NMR Spectra. Selective chemical shift changes were

observed also for several enkephalin proton resonances in360-MHz 1H NMR spectra. Certain [Met]enkephalin Phering proton resonances shifted as increments of lysoPtdChowere added (Fig. 4); resonances near 7.35 ppm moved up-field and eventually emerged from the resonance envelope(Fig. 4, spectrum E), while simultaneously resonances ini-tially near 7.32 ppm crossed these and moved downfield.§Reversing the order of addition by adding [Metlenkephalinto a solution of lysoPtdCho micelles at the ratio given in Fig.4, spectrum C, produced identical spectra. Additions oflysoPtdCho up to a 30-fold molar excess produced no spec-tral shifts beyond those observed in Fig. 4, spectrum E. Up-field movements of other Phe and Tyr proton resonances inthese spectra were also detected. A general ca. 4-fold in-

§No significant variations in chemical shifts or linewidths were ob-served for enkephalin in aromatic proton resonances at 360 MHzover the concentration range 0.35-11 mM of [Met]enkephalin in2H20 at pH - 6. This observation (and see also ref. 13) indicatedthat lipid-induced spectral changes reflect the association of pep-tide monomers with phospholipid, rather than the breakdown ofenkephalin aggregates.

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Proc. NatL Acad Sci. USA 81 (1984) 63

I B

Leu

.-- Phe

T\Gly I

1' @--<~~Ty^ .

4 5 6 0 1 2 3 4 5 6Mole ratio (lipid/peptide)

FIG. 2. Changes in positions of [Leu]enkephalin carbonyl-car-bon resonances as a function of added lysoPtdGro (A) and lysoPtd-Cho (B). Data were obtained at 90 MHz from samples and spectrasimilar to those shown in Fig. 1.

crease in linewidths vs. free [Metienkephalin accompaniedthese resonance shifts as the peptide became fully bound tolipid (i.e., Fig. 4, spectrum E). Similar trends in 1H NMRspectra were observed upon peptide binding to lysoPtdGro.

Calculation of Association Constants. Data from Figs. 2 and4 were used to estimate the association constants of [Met5]-and [Leu5]enkephalin with lysoPtdCho. The mole fraction ofpeptide bound (under the rapid exchange condition prevail-

C

S

B

1

WPhe F- Tyr

7.4 7.2 7.0 6.8ppm

FIG. 4. Regions of aromatic proton resonance in 'H NMR spec-tra (360 MHz) of [Metlenkephalin (2.5 mg in 0.5 ml of 2H20, pH6). (A) Free peptide. (B-E) Sample in spectrum A in the presence of1.0, 10.0, 20.0, and 41.0 mg of lysoPtdCho, respectively. Mole ratiosof lipid/peptide are shown.

ing for these spectra), Mf, is given by Eq. 1:

Mf =

Sobs 8free8bound 8free

[1]

where 5obs is the chemical shift of any lipid-sensitive peptideresonance at a given lipid concentration during the titration;8free is this chemical shift in free peptide; and 8bound is thechemical shift of fully-bound peptide-i.e., Fig. 4, spectrumE. The overall association constant (Ka) of peptide with lipidcan then be obtained from the expression

[2]Ka- Mf[1-Mf] [L-MfPo]'

Phe

A

IT54.5 54.0 53.5 53.0 52.5 52.0

Ppm

FIG. 3. Regions of a-carbon resonance in 13C NMR spectra (90MHz) of free [Leujenkephalin (23.5 mg in 1.5 ml of 2H20, pH = 6)(spectrum A) and the same sample after addition of one (spectrumB) and five (spectrum C) equivalents of lysoPtdGro, respectively.Mole ratios of lipid/protein are indicated. Assignments follow thosein ref. 7.

where [L] is a given lipid molar concentration and [PO] is theinitial molar concentration of peptide. Representative chemi-cal shift data were obtained from two of the Phe aromaticproton resonances (Fig. 4), and (by approximating the 6-foldmolar excess IysoPtdCho experiment as the "endpoint")from the Phe carbonyl-carbon resonance (Fig. 2B). An aver-

age value of Ka = 3.7 x 101 M-1 was calculated from valuesof Ka in Table 1.

DISCUSSIONIn addition to the electrostatic attraction between the en-

kephalin NH2-terminal amino group and (presumably) a neg-ative phosphate site of the acidic phospholipid lysoPtdGro,the present results establish site-specific hydrophobic inter-actions between [Met]- and [Leu]enkephalin both withlysoPtdGro and with the neutral lipid lysoPtdCho. To ex-

plain the physical significance of the selective chemical shiftmovement and resonance broadening observed in Figs. 1-

178.4-178.2178.0-

S0.0.

A

j\ _ Tyr

.*-Gly 3

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64 Biochemistry: Deber and Behnam

Table 1. Association constants (Ka) calculated for [Met]- and [Leu]enkephalin with lysoPtdChomicelles

Resonance* Sfreet ,oundt Sobst Mft Ka, Ml

[Met]Enkephalin 2,651.0 2,629.0 2,634.0 0.77 4.40 x 101§Phe ring protonst J 2,643.5 2,648.5 2,647.1 0.72 3.35 x 101§

[Leu]Enkephalin l 171.07 170.49 170.63 0.76 3.50 x 1o01Phe C=O carbonI*Proton chemical shifts (Hz) were taken from Fig. 4; '3C chemical shifts (ppm) were taken from Fig.2B.tSee Eq. 1 for definitions.*Ortho-, meta-, or para-ring protons; not assigned.§Calculated by Eq. 2 with [lysophosphatidylcholine] = 8.30 x 10-2 M and [PO] = 8.72 x 10-3 M.ICalculated by Eq. 2 with [lysophosphatidylcholine] = 1.12 x 10-1 M and [P0] = 2.82 x 10-2 M.

4-effects that are broadly analogous, for example, to thosenoted in the 1H NMR spectra of the aromatic (i.e., trypto-phan) region of the membrane-active protein melittin in thepresence of zwitterionic lipid micelles (14)-we postulatethat the aqueous microenvironment in selected regions of theenkephalin molecule is altered by its preferential associationwith structurally complementary regions of lipid molecules.Upon interaction with lysoPtdCho, enkephalin Gly-2, Phe,and Met/Leu carbonyl-carbon resonances show majorchemical shift changes while the positions of Gly-3 and Tyrcarbonyl-carbon resonances are virtually unchanged. Chem-ical shift data (not shown) also indicate a change in microen-vironment for the Tyr aromatic ring (A8 for Tyr t carbon =-65 Hz; AS for Tyr y carbon = +45 Hz at 8:1 lysoPtdCho/[Metlenkephalin). Because nearly identical Tyr ring shiftswere observed for peptide binding to lysoPtdGro, these re-sults suggest the involvement of the Tyr aromatic ring withnonpolar substituents of both lipids.

Location of Enkephalin in the Micelle. The line broadeningof peptide resonances observed in both 1H and '3C NMRspectra likely reflects the effective increase in molecularweight and concomitant restriction to molecular motion ofthe enkephalin bound to a lipid particle. However, theCOOH-terminal Met (Fig. 1, spectrum D) and Leu (notshown) carboxylate-carbon resonances and correspondinga-carbon resonances (Fig. 3, spectra B and C) exhibit a moresignificant increase in linewidth. That the most negative sitein the enkephalin molecule appears to be the most motional-ly restricted upon micelle binding may be explicable by itsproximity to principal sites of hydrophobic anchoring to themembrane-i.e., the Met/Leu and Phe side chains. Morelikely, because broadened resonances are frequently ob-served for carbons located near lipid particle surfaces (15,16), the enkephalin COOH-terminal residue may be incorpo-rated into the surface region of the micelle. Indeed, line-widths of lysoPtdCho and lysoPtdGro backbone carbon res-onances (data not shown) are comparable to linewidths ofresonances observed for the Met carbonyl-carbon in bound[Met]enkephalin (Fig. 1, spectrum D) and the Leu a carbonin bound [Leulenkephalin (Fig. 3, spectrum B). Thus, onecould envisage that enkephalin monomers are locatedthroughout the micelle with negative ends facing water inpositions corresponding to those of lipid phosphate groups.Taken along with chemical shift data, the overall results indi-cate the insertion or penetration of the hydrophobic enkeph-alin side chains (Met/Leu, Phe, and probably Tyr) into theinterior of the micelle.¶ These latter findings complementthose of Schwyzer et al. (ref. 18 and refs. therein), who haveused photolabeling techniques to detect hydrophobic com-

ponents of enkephalin binding to PtdCho/phosphatidic acidvesicles.

Association Constants of Enkephalin with LysoPtdCho andLysoPtdGro. The values of Ka reported in Table 1 repre-sent only the hydrophobic interactions of enkephalin tolysoPtdCho micelles. The binding of the Tyr amino group tolysoPtdGro molecules-manifested by the titration-like be-havior of the proximal Tyr carbonyl-carbon resonance (Fig.2A) between zero and one equivalent of added lysoPtdGro-is essentially complete at 1:1 lysoPtdGro/enkephalin andsignals the affinity of this group for (the lysoPtdGro) phos-phate as a counter ion (vs. ambient CF- or OH-). Althoughthis electrostatic binding to lysoPtdGro did evoke shifts thatwere absent in the lysoPtdCho experiment [e.g., in the Gly-3carbonyl-carbon resonance (Fig. 2A)], it is evident thatchemical shift movements in most resonances continue tooccur above the 1:1 lysoPtdGro/enkephalin ratio, likelyrepresenting the hydrophobic component of the overalllysoPtdGro binding to enkephalin.Measurements of Ka values from spectra of the lysoPtdGro-

enkephalin system were complicated by the dual contribu-tions of electrostatic and hydrophobic components. In oneset of calculations using the resonance of the Tyr aromatic Ccarbon-a hydrophobic interaction site that displayed paral-lel behavior with both lysoPtdCho and lysoPtdGro-valueswere obtained of Ka = 3.2 x 101 M-1 for the lysoPtdChocomplex and Ka = 5.2 x 101 M-1 for the lysoPtdGro com-plex. However, this lysoPtdGro Ka value clearly underes-timates the local attraction of the enkephalin Tyr aminogroup to lysoPtdGro; attempts to obtain Ka from the Tyr car-bonyl-carbon resonance (Fig. 2A) gave vanishingly small de-nominators in Eq. 2.

Relationship of Enkephalin/Lipid Binding to Receptors.Preparations of isolated opiate receptor proteins (19, 20)and, indeed, many membrane proteins (21, 22) appear to re-quire lipid for preservation or restoration of activity. The pu-tative role for these protein-associated lipids has been one ofensuring the maintenance of the biologically relevant proteinconformation. However, some current models of opiate re-ceptors specifically incorporate functional lipid with protein(23). In addition, several examples have been reported of lip-ids that themselves function as receptors (for a review, seeref. 24), the best characterized system being the gangliosideGM, surface membrane receptor for cholera toxin (25, 26).

Association constants (Ka) measured for enkephalin-lysoPtdCho complexes (Table 1) lead to a calculated free en-ergy of interaction AGO = - RT ln Ka = -2.3 kcal/mol forthe hydrophobic component of enkephalin-lipid binding.This value does not approach the values characteristic of en-kephalin-receptor binding [estimated from dissociation con-stants that are in the nanomolar range (see for example ref.27) to be = -12 kcal/mol]. Nevertheless, the AGO we ob-tained is comparable to the energy of interaction of 3 kcal/mol deduced by Berg and Purcell (28) to be required for ad-sorption of a molecule to a membrane surface and its two-

9Based on lipid/peptide ratios required to bind peptide fully and thefact that 150-200 lipid monomers comprise an average lysoPtdChomicelle (6, 17), we estimated that 10 enkephalin molecules would bethus dispersed in a given micelle under the experimental conditionsof Fig. 4, spectrum D.

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dimensional diffusion toward a cell receptor. Also, thelysoPtdCho AGO value does not include the potentially great-er contribution from electrostatic binding (see below). Thus,the present results provide experimental confirmation for thefunctioning of a phospholipid surface as an enkephalin ad-sorbent.

Conformation of Enkephalin in the Lipid Environment. Therelationship of peptide conformational features in solution tothose in the environment of the membrane receptor is one ofconsiderable importance for deduction of hormone struc-ture-activity relationships (29), particularly when viewed ina context where the conformation of the protein receptor isconsidered also to be heavily influenced by surrounding lip-ids. In fact, peptide/protein binding to membrane prepara-tions is often reported [i.e., from circular dichroism mea-surements (30)] to be accompanied by alterations in grosspeptide conformation. In the present NMR spectra, selectivechemical shift and linewidth changes take place throughoutthe 360-MHz 1H NMR spectra of the enkephalins upon mi-celle binding (e.g., Fig. 4), indicative of specificity or sided-ness for the mode of attachment of the peptide to the micelle.However, in preliminary 1H NMR studies of all enkephalinresonances not obscured by lipid resonances (i.e., Phe, Metand Tyr P-proton resonances; Met y-proton resonances; andthe Phe a-proton resonance) (31), no significant variationswere found in vicinal coupling constants, as might be expect-ed to accompany major redistribution of side-chain rotamerpopulations on lipid binding. (Note that these data provideno direct information concerning the peptide backbone.) So-lution conformation(s) that have been proposed for enkepha-lin, such as those containing a folded, intramolecularly H-bonded /8-turn structure centered around Gly-3 (32-34),could thus be largely maintained upon micelle binding; suchstructures do appear to place enkephalin hydrophobic sub-stituents on a nonpolar face suitable for lipid interaction.Role of Lipids in Enkephalin Binding. We conclude that the

major influences of lipid reside in (0) binding the hormonethrough a combination of electrostatic attractions and hydro-phobic interactions, thereby (it) reducing its rates of localmolecular motions and (iii) limiting the degrees offreedom ofindividual peptide residues with respect to their position andorientation in the membrane. The finding that the enkephalinCOOH terminus is located in the membrane surface providesan illustration of the combined consequences of effects i-iii.Although the overall enkephalin-lipid association may berelatively nonspecific (membranes adsorb a variety of sub-stances), a further role for lipid in peptide hormone actionmay be one of concentrating the hormone into the membranesuch that the effective concentration of the hormone in themicroenvironment of the receptor is higher than in the bulksolution.The greater strength of electrostatic vs. hydrophobic

forces and the preponderance of negatively charged mem-brane lipids encountered by enkephalins in vivo (phosphati-dylserine, cerebroside sulfate) suggests to us an attraction-interaction model for the binding of enkephalin to phospho-lipids. In such a model, electrostatic forces could represent anecessary condition for activity [i.e., the nucleation step inthe "zipper" model of Burgen et al. (35)], while site-specifichydrophobic binding of lipids to strategic residues in the pep-tide hormone could modulate the propensity for hormonetransfer to the membrane phase and conceivably aid in influ-encing the specific orientation of the peptide as it diffusestoward its receptor protein.

We thank Prof. Robert Schwyzer (Eidgenossiche TechnischeHochschule, Zurich) for communicating results from his laboratoryto us prior to publication and Dr. Vincent Madison (Hoffmann-LaRoche, Inc., Nutley, NJ) for a stimulating discussion. NMR spectrawere recorded at the Toronto Biomedical NMR Center, which is

supported in part by the Medical Research Council of Canada underMaintenance Grant MT-6499. This work was supported in part bygrants to C.M.D. from the Medical Research Council (MT-5810) andthe Multiple Sclerosis Society of Canada.

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