Proc. Nati. Acad. Sci. USA Vol. 81, pp. 61-65, January 1984 Biochemistry
Role of membrane lipids in peptide hormone function: Binding of enkephalins 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, Physicochemical studies of the association of materials peptide hormones must be transferred from an aqueous phase such as insulin (2), glucagon (3), and the apolipoproteins (4) to the lipid-rich environment of their membrane-bound recep- with membrane preparations in vitro have been performed to tor proteins. We have investigated the possible influence of determine the specific manner through which phospholipids phospholipids in this process, using 360-MHz 'H and 90-MHz contribute to their functioning in vivo. Enkephalin itself has 13C NMR spectroscopy to examine the association of the opioid been shown to associate through ionic attraction to negative peptides [Met]- and [Leu]enkephalins (Tyr-Gly-Gly-Phe-Met/ lipids such as phosphatidylserine (5). Using high-resolution Leu) with phospholipid micelles. Binding of peptides to lipid 1H and 13C NMR techniques, we have now obtained evi- was monitored in NMR spectra by selective chemical shift dence for hydrophobic interaction of two endogenous 1 2 3 movements (e.g., the Phe aromatic ring protons) and residue- opioid peptides, [Met]- and [Leu]enkephalin (Tyr-Gly-Gly- specific line broadening (e.g., of Met/Leu carbonyl- and a- 4 5 carbon resonances). Results established that the zwitterionic Phe-Met/Leu), with a neutral phospholipid, lysophosphati- hormones associate hydrophobically both with a neutral lipid dylcholine (lysoPtdCho). The peptides also interact through (lysophosphatidylcholine) and (also electrostatically) with a a combination of electrostatic and hydrophobic interactions negative lipid (lysophosphatidylglycerol). An association coh- with the anionic lipid lysophosphatidylglycerol (lysoPtd- stant of Ka = 3.7 x 101 M-1 was calculated for the hydropho- Gro). In addition, we have been able to infer specific sites on bic binding of enkephalin to lysophosphatidylcholine. NMR the peptide of major interaction with lipid and to obtain a data suggested that enkephalin binds to the lipid with Met/ quantitative measure of the hydrophobic component of pep- Led, Phe, and likely Tyr side-chain substituents associated tide-lipid interaction. The possible role(s) of endogenous with nonpolar interior regions of the micelle, whereas the phospholipids is evaluated on the basis of these data. COOH-terminal carboxylate moiety of the peptide is located in the surface of the lipid particle. An "attraction-interaction" MATERIALS AND METHODS model is proposed for hormone-lipid association wherein neg- [Met]Enkephalin (Tyr-Gly-Gly-Phe-Met; Bachem Fine ative lipids attract the hormone electrostatically, while site- Chemicals, Torrance, CA), [Leulenkephalin (Tyr-Gly-Gly- specific hydrophobic contacts facilitate its entry, concentra- Phe-Leu; Fluka, Hauppauge, NY), egg L-a-lysoPtdCho (Sig- tion, and orientation into the lipid phase. ma), L-a-lysoPtd-DL-Gro (Sigma), and praseodymium nitrate pentahydrate (99.9%; Alfa-Ventron, Danvers, MA) were Aqueous-soluble proteins and peptide hormones are often used without further purification. bound to or transferred into membranes in conjunction with NMR samples were prepared with a peptide concentration their biological functions. Because a hormone as well as its of 8.72 mM for 1H NMR and from 27.3 to 28.2 mM for 13C membrane-embedded receptor protein contain potential NMR studies. Lipid concentrations, given in figure legends, sites of association with lipids, one may hypothesize that were generally above critical micelle concentration levels surrounding endogenous phospholipids could potentially (6). The quoted pH values are pH meter readings in 2H20 play any of several roles in mediating the transfer. These (Merck Sharp and Dohme, Montreal; 99.8%) that were un- could include: facilitating the capture, entry, and concentra- corrected for the deuterium isotope effect and were mea- tion of the aqueous-soluble hormone or neurotransmitter sured directly in the NMR tubes at room temperature. into the microenvironment of the receptor; orienting the pep- 1H NMR spectra were determined at 360 MHz with the tide in the membrane vis-d-vis the receptor by restricting mo- Nicolet NIC spectrometer operating in the Fourier transform lecular motions; and/or, in a more specific function, con- mode with 16,000 data points and typically 250 accumula- verting the hormone into a conformation required for elicit- tions for each spectrum. A 5-sec frequency pulse was used to ing biological activity. suppress the residual 1H2HO resonance. Temperature was These circumstances are relevant to the enkephalins-the 23 ± 10C. Chemical shifts are given in ppm after standardiza- peptide neurotransmitters that compete with morphine and tion of the spectrometer to external tetramethylsilane. Lipid- its derivatives for opiate binding sites in the brain (1). To induced shifts were measured after introducing successive, examine the above possibilities, we have initiated a study of weighed amounts of the lipid into the 2H20 solution of pep- some enkephalin-lipid complexes to determine (i) whether tide. hydrophobic interactions as well as electrostatic attractions 'H-decoupled 13C NMR spectra were determined at 90 contribute significantly to their stabilization, particularly in MHz with the Nicolet NIC spectrometer operating in the complexes with zwitterionic (net neutral) lipids, and (ii) the Fourier transform mode with 16,000 data points and typical- extent to which these interactions influence hormone mo- ly 12,000 accumulations for each spectrum. Chemical shifts tional and conformational parameters. are reported in ppm downfield from internal [2-'3C]acetoni- trile reference standard. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: PtdCho, phosphatidylcholine; lysoPtdCho, lyso- in accordance with 18 U.S.C. §1734 solely to indicate this fact. phosphatidylcholine; lysoPtdGro, lysophosphatidylglycerol.
61 Downloaded by guest on September 26, 2021 62 Biochemistry: Deber and Behnam Proc. NatL Acad ScL USA 81 (1984) 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 were introduced into a solution of the peptide in 2H20. RESULTS 13C 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 valuable tools for investigation of protein-membrane interactions (9- 12). Significant upfield chemical shift changes (A8) were ob- B. +35 mg served for three of the five enkephalin carbonyl carbons lysoPtdCho (Gly-2, Phe, and Met), while the NH2-terminal Tyr and Gly-3 carbonyl-carbon resonances displayed little or no change up Wet l'dE < to approximately 5-fold molar excess of lipid (Fig. 1, spec- trum D).t Binding of [Leu]enkephalin to lysoPtdCho micelles pro- A duced similar results (Fig. 2B), whereas the chemical shift Phe changes observed for the titration of [Leu]enkephalin with micelles prepared from the negatively charged lipid lysoPtdGro are shown in Fig. 2A. In the latter experiment, the chemical shift of the NH2-terminal Tyr carbonyl-carbon resonance shifted downfield from 169.1 to 169.9 ppm after addition of the equivalent amount of lysoPtdGro. This be- havior should be contrasted with the negligible chemical 177 175 173 171 169 shift change for Tyr carbonyl-carbon resonances of both PPm [Met]- and [Leu]enkephalin over the range of 0 to 6-fold mo- lar excess of lysoPtdCho (Fig. 1, spectrum D and Fig. 2B). It FIG. 1. Regions of carbonyl-carbon resonances in 13C NMR is emphasized that throughout all additions of lipids, the pH spectra (90 MHz) of [Met]enkephalin (23.5 mg in 1.5 ml of 2H20, pH remained constant ±0.1 unit. The Gly-3 carbonyl-carbon - 6) (spectrum A) to which increasing portions of lysoPtdCho have resonance also reflected the "titration" behavior between 0 been added (spectra B-D). Mole ratios of lipid/peptide range from ca. 2 in spectrum B to ca. 6 in spectrum D. Resonances offree pep- and 1:1 molar ratio of lysoPtdGro/peptide (Fig. 2A) vs. its tide (spectrum A) were assigned in accordance with Khaled et al. relative insensitivity to added lysoPtdCho (Fig. 2B). An (ref. 7; see also footnote t). Numbers in spectrum D indicate the overall comparison of the trends above 1:1 lysoPtdGro/ total chemical shift changes (Hz) from spectrum A to spectrum D; peptide in Fig. 2A with Fig. 2B suggests that the upfield positive shifts are upfield. _L, ester carbonyl-carbon resonance of movements of Leu, Phe, and Gly-2 carbonyl-carbon reso- lysoPtdCho. nances are comparable, indicating that the enkephalin-lipid interactions manifested by these shifts are taking place in a and lysoPtdGro. Similar phenomena are observed in the a- corresponding manner with both lipids, being masked, in ef- carbon region of micelle-bound peptides (Fig. 3); for exam- fect, by the initial primary attraction apparent in the lysoPtd- ple, the [Leu]enkephalin a-carbon resonance is selectively Gro system. broadened in the presence of lysoPtdGro [linewidth = 3.5 The chemical shift changes upon binding lysoPtdCho mi- Hz for free [Leu]enkephalin (Fig. 3, spectrum A) and 10.5 celles are accompanied by general line broadening (Fig. 1, Hz at 1:1 lysoPtdGro/peptide (Fig. 3, spectrum B)]. spectra A-D), but additional selective line broadening clear- 'H NMR Spectra. Selective chemical shift changes were ly occurs for the COOH-terminal Met carboxylate-carbon observed also for several enkephalin proton resonances in resonances [linewidth (uncorrected) = 3.3 Hz (Fig. 1, spec- 360-MHz 1H NMR spectra. Certain [Met]enkephalin Phe trum A) for the free [Met]enkephalin and 9.1 Hz for bound ring proton resonances shifted as increments of lysoPtdCho peptide (Fig. 1, spectrum D)]. Parallel effects are observed were added (Fig. 4); resonances near 7.35 ppm moved up- for both [Met]- and [Leu]enkephalin with both lysoPtdCho field and eventually emerged from the resonance envelope (Fig. 4, spectrum E), while simultaneously resonances ini- tThe two resonances designated in Fig. 1 as Gly-2 and Gly-3 corre- tially near 7.32 ppm crossed these and moved downfield.§ spond to assignments inferred (7) from a series of 13C NMR spectra Reversing the order of addition by adding [Metlenkephalin of enkephalins and several analog peptides. We have obtained addi- tional support for these assignments from a study of effects on 13C to a solution of lysoPtdCho micelles at the ratio given in Fig. NMR spectra of [Met]- and [Leu]enkephalin in aqueous solution 4, spectrum C, produced identical spectra. Additions of upon binding to paramagnetic praseodymium ions. In the presence lysoPtdCho up to a 30-fold molar excess produced no spec- of ca. 5-fold excess Pr(NO3)3, the resonance labeled Gly-3 moved tral shifts beyond those observed in Fig. 4, spectrum E. Up- 0.56 ppm downfield, vs. a change of 0.33 ppm for that labeled Gly- field movements of other Phe and Tyr proton resonances in 2, as expected for decreasing proximity to Pr3+ bound at the these spectra were also [Met]enkephalin COOH-terminal carboxylate group. Parallel detected. A general ca. 4-fold in- trends were observed for the pair of Gly a-carbon resonances; the upfield resonance shifted 0.22 ppm, while the downfield resonance §No significant variations in chemical shifts or linewidths were ob- was unaffected. This behavior is in accord with expectations from served for enkephalin in aromatic proton resonances at 360 MHz the absolute assignments of these resonances to Gly-3 and Gly-2 a- over the concentration range 0.35-11 mM of [Met]enkephalin in carbons, respectively, obtained from the NMR spectra of 13C-en- 2H20 at pH - 6. This observation (and see also ref. 13) indicated riched synthetic [Met]enkephalin samples (5) (see also ref. 8). that lipid-induced spectral changes reflect the association of pep- Spectra of [Leu]enkephalin and Pr3+ showed qualitatively parallel tide monomers with phospholipid, rather than the breakdown of shifts. enkephalin aggregates. Downloaded by guest on September 26, 2021 Biochemistry: Deber and Behnam Proc. NatL Acad Sci. USA 81 (1984) 63
178.4- A I B 178.2 178.0- Leu
S 0. .-- Phe 0.
j\ _ Tyr T\Gly I
1' @--<~~Ty .*-Gly 3 ^ . 4 5 6 0 1 2 3 4 5 6 Mole 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 spectra similar to those shown in Fig. 1.
crease in linewidths vs. free [Metienkephalin accompanied these resonance shifts as the peptide became fully bound to WPhe F- lipid (i.e., Fig. 4, spectrum E). Similar trends in 1H NMR Tyr spectra were observed upon peptide binding to lysoPtdGro. Calculation of Association Constants. Data from Figs. 2 and 7.4 7.2 7.0 6.8 4 were used to estimate the association constants of [Met5]- ppm and [Leu5]enkephalin with lysoPtdCho. The mole fraction of FIG. 4. Regions of aromatic proton resonance in 'H NMR spec- peptide bound (under the rapid exchange condition prevail- tra (360 MHz) of [Metlenkephalin (2.5 mg in 0.5 ml of 2H20, pH 6). (A) Free peptide. (B-E) Sample in spectrum A in the presence of C 1.0, 10.0, 20.0, and 41.0 mg oflysoPtdCho, respectively. Mole ratios of lipid/peptide are shown. ing for these spectra), Mf, is given by Eq. 1:
S Sobs Mf = 8free [1] 8bound 8free
where 5obs is the chemical shift of any lipid-sensitive peptide resonance at a given lipid concentration during the titration; B 8free is this chemical shift in free peptide; and 8bound is the chemical shift of fully-bound peptide-i.e., Fig. 4, spectrum E. The overall association constant (Ka) of peptide with lipid can then be obtained from the expression 1 Ka- Mf [2] [1-Mf] [L-MfPo]' Phe where [L] is a given lipid molar concentration and [PO] is the initial molar concentration ofpeptide. Representative chemi- A cal shift data were obtained from two of the Phe aromatic proton resonances (Fig. 4), and (by approximating the 6-fold molar 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 values of Ka in Table 1.
IT DISCUSSION 54.5 54.0 53.5 53.0 52.5 52.0 In addition to the electrostatic attraction between the en- Ppm kephalin NH2-terminal amino group and (presumably) a neg- ative phosphate site of the acidic phospholipid lysoPtdGro, FIG. 3. Regions of a-carbon resonance in 13C NMR spectra (90 the present results establish site-specific hydrophobic inter- MHz) of free [Leujenkephalin (23.5 mg in 1.5 ml of 2H20, pH = 6) actions between [Met]- and both with (spectrum A) and the same sample after addition of one (spectrum [Leu]enkephalin ex- B) and five (spectrum C) equivalents of lysoPtdGro, respectively. lysoPtdGro and with the neutral lipid lysoPtdCho. To Mole ratios of lipid/protein are indicated. Assignments follow those plain the physical significance of the selective chemical shift in ref. 7. movement and resonance broadening observed in Figs. 1- Downloaded by guest on September 26, 2021 64 Biochemistry: Deber and Behnam Proc. Nad Acad Sci. USA 81 (1984)
Table 1. Association constants (Ka) calculated for [Met]- and [Leu]enkephalin with lysoPtdCho micelles 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 1o01 Phe 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 those ponents of enkephalin binding to PtdCho/phosphatidic acid noted in the 1H NMR spectra of the aromatic (i.e., trypto- vesicles. phan) region of the membrane-active protein melittin in the Association Constants of Enkephalin with LysoPtdCho and presence of zwitterionic lipid micelles (14)-we postulate LysoPtdGro. The values of Ka reported in Table 1 repre- that the aqueous microenvironment in selected regions of the sent only the hydrophobic interactions of enkephalin to enkephalin molecule is altered by its preferential association lysoPtdCho micelles. The binding of the Tyr amino group to with structurally complementary regions of lipid molecules. lysoPtdGro molecules-manifested by the titration-like be- Upon interaction with lysoPtdCho, enkephalin Gly-2, Phe, havior of the proximal Tyr carbonyl-carbon resonance (Fig. and Met/Leu carbonyl-carbon resonances show major 2A) between zero and one equivalent of added lysoPtdGro- chemical shift changes while the positions of Gly-3 and Tyr is essentially complete at 1:1 lysoPtdGro/enkephalin and carbonyl-carbon resonances are virtually unchanged. Chem- signals the affinity of this group for (the lysoPtdGro) phos- ical shift data (not shown) also indicate a change in microen- phate as a counter ion (vs. ambient CF- or OH-). Although vironment for the Tyr aromatic ring (A8 for Tyr t carbon = this electrostatic binding to lysoPtdGro did evoke shifts that -65 Hz; AS for Tyr y carbon = +45 Hz at 8:1 lysoPtdCho/ were absent in the lysoPtdCho experiment [e.g., in the Gly-3 [Metlenkephalin). Because nearly identical Tyr ring shifts carbonyl-carbon resonance (Fig. 2A)], it is evident that were observed for peptide binding to lysoPtdGro, these re- chemical shift movements in most resonances continue to sults suggest the involvement of the Tyr aromatic ring with occur above the 1:1 lysoPtdGro/enkephalin ratio, likely nonpolar substituents of both lipids. representing the hydrophobic component of the overall Location of Enkephalin in the Micelle. The line broadening lysoPtdGro binding to enkephalin. of peptide resonances observed in both 1H and '3C NMR Measurements of Ka values from spectra of the lysoPtdGro- spectra likely reflects the effective increase in molecular enkephalin system were complicated by the dual contribu- weight and concomitant restriction to molecular motion of tions of electrostatic and hydrophobic components. In one the enkephalin bound to a lipid particle. However, the set of calculations using the resonance of the Tyr aromatic C COOH-terminal Met (Fig. 1, spectrum D) and Leu (not carbon-a hydrophobic interaction site that displayed paral- shown) carboxylate-carbon resonances and corresponding lel behavior with both lysoPtdCho and lysoPtdGro-values a-carbon resonances (Fig. 3, spectra B and C) exhibit a more were obtained of Ka = 3.2 x 101 M-1 for the lysoPtdCho significant increase in linewidth. That the most negative site complex and Ka = 5.2 x 101 M-1 for the lysoPtdGro com- in the enkephalin molecule appears to be the most motional- plex. However, this lysoPtdGro Ka value clearly underes- ly restricted upon micelle binding may be explicable by its timates the local attraction of the enkephalin Tyr amino proximity to principal sites of hydrophobic anchoring to the group to lysoPtdGro; attempts to obtain Ka from the Tyr car- membrane-i.e., the Met/Leu and Phe side chains. More bonyl-carbon resonance (Fig. 2A) gave vanishingly small de- likely, because broadened resonances are frequently ob- nominators in Eq. 2. served for carbons located near lipid particle surfaces (15, Relationship of Enkephalin/Lipid Binding to Receptors. 16), the enkephalin COOH-terminal residue may be incorpo- Preparations of isolated opiate receptor proteins (19, 20) rated into the surface region of the micelle. Indeed, line- and, indeed, many membrane proteins (21, 22) appear to re- widths of lysoPtdCho and lysoPtdGro backbone carbon res- quire lipid for preservation or restoration of activity. The pu- onances (data not shown) are comparable to linewidths of tative role for these protein-associated lipids has been one of resonances observed for the Met carbonyl-carbon in bound ensuring the maintenance of the biologically relevant protein [Met]enkephalin (Fig. 1, spectrum D) and the Leu a carbon conformation. However, some current models of opiate re- in bound [Leulenkephalin (Fig. 3, spectrum B). Thus, one ceptors specifically incorporate functional lipid with protein could envisage that enkephalin monomers are located (23). In addition, several examples have been reported of lip- throughout the micelle with negative ends facing water in ids that themselves function as receptors (for a review, see positions corresponding to those of lipid phosphate groups. ref. 24), the best characterized system being the ganglioside Taken along with chemical shift data, the overall results indi- GM, surface membrane receptor for cholera toxin (25, 26). cate the insertion or penetration of the hydrophobic enkeph- Association constants (Ka) measured for enkephalin- alin side chains (Met/Leu, Phe, and probably Tyr) into the lysoPtdCho complexes (Table 1) lead to a calculated free en- interior of the micelle.¶ These latter findings complement ergy of interaction AGO = - RT ln Ka = -2.3 kcal/mol for those of Schwyzer et al. (ref. 18 and refs. therein), who have the hydrophobic component of enkephalin-lipid binding. used photolabeling techniques to detect hydrophobic com- 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. 9Based on lipid/peptide ratios required to bind peptide fully and the = we fact that 150-200 lipid monomers comprise an average lysoPtdCho 27) to be -12 kcal/mol]. Nevertheless, the AGO ob- micelle (6, 17), we estimated that 10 enkephalin molecules would be tained is comparable to the energy of interaction of 3 kcal/ thus dispersed in a given micelle under the experimental conditions mol deduced by Berg and Purcell (28) to be required for ad- of Fig. 4, spectrum D. sorption of a molecule to a membrane surface and its two- Downloaded by guest on September 26, 2021 Biochemistry: Deber and Behnam Proc. NatL Acad ScL USA 81 (1984) 65
dimensional diffusion toward a cell receptor. Also, the supported in part by the Medical Research Council of Canada under lysoPtdCho AGO value does not include the potentially great- Maintenance Grant MT-6499. This work was supported in part by er contribution from electrostatic binding (see below). Thus, grants to C.M.D. from the Medical Research Council (MT-5810) and the present results provide experimental confirmation for the the Multiple Sclerosis Society of Canada. functioning of a phospholipid surface as an enkephalin ad- sorbent. 1. Kosterlitz, H. W. & Hughes, J. (1975) Life Sci. 17, 91-96. Conformation of Enkephalin in the Lipid Environment. The 2. Wu, C.-S. & Yang, J. T. (1981) Biochim. Biophys. Acta 667, relationship of peptide conformational features in solution to 285-293. those in the environment of the membrane receptor is one of 3. Bosch, C., Brown, L. R. & Wuthrich, K. (1980) Biochim. considerable importance for deduction of hormone struc- Biophys. Acta 603, 298-312. ture-activity relationships (29), particularly when viewed in 4. Massey, J. B., Gotto, A. M., Jr., & Pownall, H. J. (1980) J. a context where the conformation of the protein receptor is Biol. Chem. 255, 10167-10173. 5. Jarrell, H. C., Deslauriers, R., McGregor, W. H. & Smith, considered also to be heavily influenced by surrounding lip- I. C. P. (1980) Biochemistry 19, 385-390. ids. In fact, peptide/protein binding to membrane prepara- 6. Kellaway, I. W. & Saunders, L. (1970) Chem. Phys. Lipids 4, tions is often reported [i.e., from circular dichroism mea- 261-268. surements (30)] to be accompanied by alterations in gross 7. Khaled, M. A., Urry, D. W. & Bradley, R. J. (1979) J. Chem. peptide conformation. In the present NMR spectra, selective Soc. Perkin Trans. 2, 1693-1699. chemical shift and linewidth changes take place throughout 8. Stimson, E. R., Meinwald, Y. C. & Scheraga, H. A. (1979) the 360-MHz 1H NMR spectra of the enkephalins upon mi- Biochemistry 18, 1661-1671. celle binding (e.g., Fig. 4), indicative of specificity or sided- 9. Hagen, D. S., Weiner, J. H. & Sykes, B. D. (1979) Biochemis- ness for the mode of attachment of the peptide to the micelle. try 18, 2007-2012. 10. Brown, L. R. (1979) Biochim. Biophys. Acta 557, 135-148. However, in preliminary 1H NMR studies of all enkephalin 11. Hughes, D. W., Stollery, J. G., Moscarello, M. A. & Deber, resonances not obscured by lipid resonances (i.e., Phe, Met C. M. (1982) J. Biol. Chem. 257, 4698-4700. and Tyr P-proton resonances; Met y-proton resonances; and 12. Gierasch, L. M., Lacy, J. E., Thompson, K. F., Rockwell, the Phe a-proton resonance) (31), no significant variations A. L. & Watnick, P. I. (1982) Biophys. J. 37, 275-284. were found in vicinal coupling constants, as might be expect- 13. Tancrede, P., Deslauriers, R., McGregor, W. H., Ralston, E., ed to accompany major redistribution of side-chain rotamer Sarantakis, D., Somorjai, R. L. & Smith, I. C. P. (1978) Bio- populations on lipid binding. (Note that these data provide chemistry 17, 2905-2914. no direct information concerning the peptide backbone.) So- 14. Lauterwein, J., Bosch, C., Brown, L. R. & Wuthrich, K. lution conformation(s) that have been proposed for enkepha- (1979) Biochim. Biophys. Acta 556, 244-264. 15. Godici, P. E. & Landsberger, F. R. (1974) Biochemistry 13, lin, such as those containing a folded, intramolecularly H- 362-368. bonded /8-turn structure centered around Gly-3 (32-34), 16. Levine, Y. K., Birdsall, N. J. M., Lee, A. G. & Metcalfe, could thus be largely maintained upon micelle binding; such J. C. (1972) Biochemistry 11, 1416-1421. structures do appear to place enkephalin hydrophobic sub- 17. Smith, R. & McDonald, B. J. (1979) Biochim. Biophys. Acta stituents on a nonpolar face suitable for lipid interaction. 554, 133-147. Role of Lipids in Enkephalin Binding. We conclude that the 18. Schwyzer, R., Gremlich, H.-U., Gysin, B. & Fringeli, U.-P. major influences of lipid reside in (0) binding the hormone (1983) in Peptides, 1982, Proceedings of 17th European Peptide through a combination of electrostatic attractions and hydro- Symposium, eds. Blaha, K. & Malon, P. (DeGruyter, Berlin), phobic interactions, thereby reducing its rates of local in press. (it) 19. Abood, L. G. & Takeda, F. (1976) Eur. J. Pharmacol. 39, 71- molecular motions and (iii) limiting the degrees offreedom of 74. individual peptide residues with respect to their position and 20. Abood, L. G., Salem, N., MacNeil, M. & Butler, M. (1978) orientation in the membrane. The finding that the enkephalin Biochim. Biophys. Acta 530, 35-46. COOH terminus is located in the membrane surface provides 21. Klausner, R. D., Bridges, K., Tsunoo, H., Blumenthal, R., an illustration of the combined consequences of effects i-iii. Weinstein, J. N. & Ashwell, G. (1980) Proc. Nati. Acad. Sci. Although the overall enkephalin-lipid association may be USA 77, 5087-5091. relatively nonspecific (membranes adsorb a variety of sub- 22. Hebdon, G. M., LeVine, H., III, Sahyoun, N. E., Schmitges, stances), a further role for lipid in peptide hormone action C. J. & Cuatrecasas, P. (1981) Proc. Nati. Acad. Sci. USA 78, may be one of concentrating the hormone into the membrane 120-123. 23. Lee, N. M. & Smith, A. P. (1980) Life Sci. 26, 1459-1464. such that the effective concentration of the hormone in the 24. Hakomori, S. (1981) Annu. Rev. Biochem. 50, 733-764. microenvironment of the receptor is higher than in the bulk 25. Fishman, P. H. & Brady, R. 0. (1976) Science 194, 906-915. solution. 26. Brady, R. P. & Fishman, P. H. (1979) Adv. Enzymol. 50, 303- The greater strength of electrostatic vs. hydrophobic 323. forces and the preponderance of negatively charged mem- 27. Simonds, W. F., Koski, G., Streaty, R. A., Hjelmeland, L. M. brane lipids encountered by enkephalins in vivo (phosphati- & Klee, W. A. (1980) Proc. Nati. Acad. Sci. USA 77, 4623- dylserine, cerebroside sulfate) suggests to us an attraction- 4627. interaction model for the binding of enkephalin to phospho- 28. Berg, H. C. & Purcell, E. M. (1977) Biophys. J. 20, 193-219. lipids. In such a model, electrostatic forces could represent a 29. Hruby, V. J. (1982) Life Sci. 31, 189-199. 30. Wu, C.-S. C., Ikeda, K. & Yang, J. T. (1981) Biochemistry 20, necessary condition for activity [i.e., the nucleation step in 566-570. the "zipper" model of Burgen et al. (35)], while site-specific 31. Behnam, B. A. & Deber, C. M. (1983) in Peptides: Structure hydrophobic binding of lipids to strategic residues in the pep- and Function, Proceedings of the Eighth American Peptide tide hormone could modulate the propensity for hormone Symposium, eds. Hruby, V. J. & Rich, D. (Pierce Chemical, transfer to the membrane phase and conceivably aid in influ- Rockford, IL), in press. encing the specific orientation of the peptide as it diffuses 32. Garbay-Jaureguiberry, C., Roques, B. P., Oberlin, R., An- toward its receptor protein. teunis, M. & Lala, A. K. (1976) Biochem. Biophys. Res. Com- mun. 71, 558-565. We thank Prof. Robert Schwyzer (Eidgenossiche Technische 33. Jones, C. R., Gibbons, W. A. & Garsky, V. (1976) Nature Hochschule, Zurich) for communicating results from his laboratory (London) 262, 779-782. to us prior to publication and Dr. Vincent Madison (Hoffmann-La 34. Zetta, L. & Cabassi, F. (1982) Eur. J. Biochem. 122, 215-222. Roche, Inc., Nutley, NJ) for a stimulating discussion. NMR spectra 35. Burgen, A. S. V., Roberts, G. C. K. & Feeney, J. (1975) Na- were recorded at the Toronto Biomedical NMR Center, which is ture (London) 253, 753-755. Downloaded by guest on September 26, 2021