13C NMR Method for the Determination of Peptide and Protein

The natural abundance 13C NMR method was applied to directly determine the binding site of peptides and proteins in lipid bilayers and emulsions on th...
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J. Phys. Chem. B 2001, 105, 12616-12621

13C

NMR Method for the Determination of Peptide and Protein Binding Sites in Lipid Bilayers and Emulsions Emiko Okamura,* Tomohiro Kimura, and Masaru Nakahara* Institute for Chemical Research, Kyoto UniVersity, Uji, Kyoto 611-0011, Japan

Masafumi Tanaka and Tetsurou Handa Graduate School of Pharmaceutical Sciences, Kyoto UniVersity, Sakyo-ku, Kyoto 606-8501, Japan

Hiroyuki Saito Osaka Branch, National Institute of Health Sciences, Osaka 540-0006, Japan ReceiVed: July 9, 2001; In Final Form: September 17, 2001

The natural abundance 13C NMR method was applied to directly determine the binding site of peptides and proteins in lipid bilayers and emulsions on the atomic level. Reliable NMR criteria for the location and depth of peptides and proteins in membranes were shown by the chemical shift and line width analyses, which reproduced not only the deep penetration of a transmembrane channel peptide gramicidin A but also the superficial binding of Ac-18A-NH2 (Ac-DWLKAFYDKVAEKLKEAF-NH2), a synthetic model peptide of amphipathic helices of plasma apolipoprotein A-I (apoA-I). The reliability was ensured by the NMR information, which was consistent with the recent X-ray diffraction study of Ac-18A-NH2 in oriented lipid bilayers (Hristova et al. J. Mol. Biol. 1999, 290, 99). Our method first provided the atomic-level evidence for native apoA-I binding in egg phosphatidylcholine (EPC) vesicles and triolein (TO)-EPC emulsions as spherical model lipoproteins. Membrane perturbation was most significant at EPC glycerol and ester carbonyl sites when apoA-I was bound to EPC small unilamellar vesicles. This indicates not deep but shallow penetration of apoA-I into the membrane interface whose polarity is intermediate between water and the hydrophobic core. The binding preference for the interfacial site of membranes was confirmed by the common binding site between apoA-I and its model peptide Ac-18A-NH2. Membrane structural modulation by apoA-I was, however, moderate at the bilayer headgroup and the alkyl chain region near the interface. The shallow penetration of apoA-I was also found in TO-EPC emulsions, a protein-free model of triglyceride-rich lipoproteins (chylomicrons) in plasma.

Introduction Drug delivery (DD) into lipid bilayer membranes is of importance as a primary step of the biological action. To elucidate the DD mechanism, the molecular information about the binding site of drugs in membranes is essential. Bioactive peptides and proteins are kinds of drugs. For a better understanding of a wide variety of drug activities, from small molecules to macromolecular proteins, the establishment of the general concepts and method for determining drug location and binding site in membranes is of great significance. NMR probing of the specific site of membranes is a potential method for the determination of protein location and depth in membranes on the molecular level. For the distinguishability of membrane atom sites with high resolution, the solution NMR is particularly informative. The application of the method to hydrated lipid bilayers can be powerful to specify the location of membrane proteins on the atomic level. The information is valuable because the application has been so far limited to the micellar system. Recent progress in high-resolution 13C NMR has enabled us to distinguish individual carbon atom site of lipid * To whom correspondence should be addressed. Phone: +81-774-383074, Fax: +81-774-38-3076, E-mail: [email protected]. E-mail: [email protected].

bilayer membranes from polar headgroup to apolar chains.1 We have distinguished the location and orientation of drugs such as alkylbenzenes, benzyl alcohol, and bisphenol A which are solubilized in phospholipid bilayer vesicles without 13C enrichment.2,3 In this paper, we report an extensive application of the natural abundance 13C NMR to specify protein (peptide) binding sites in lipid bilayers and emulsions. We have first shown that NMR and X-ray diffraction results have the identical evidence for the binding site of an amphipathic R-helical peptide Ac-18A-NH2 (Ac-DWLKAFYDKVAEKLKEAF-NH2) in bilayers. The NMR method has been also applied to the native apolipoprotein A-I (apoA-I) binding in bilayers and emulsions; the direct atom-level information has not been available yet. Location and depth of proteins (peptides) in membranes have been the subject of special interest in relation to biological functions. The X-ray diffraction method gives us precise information about the structure and transbilayer location of peptides with atomic resolution.4-6 The application requires diffractable periodical structures, such as oriented multilamellar membranes, and the information has not been available yet for native membrane proteins. Almost no limitations are present in the NMR application. The depth in membranes has been most recently discussed by Prosser et al.7,8 by using 19F NMR of fluorinelabeled surfactants and peptides, which are incorporated into

10.1021/jp012583k CCC: $20.00 © 2001 American Chemical Society Published on Web 11/28/2001

Determination of Peptide and Protein Binding Sites lipid bilayer model membranes. They have suggested the possibility of the application of 19F NMR to larger membraneassociated molecules. Here we propose the natural abundance 13C NMR as a simple but general method for the reliable determination of protein location and depth in membranes without labeling. To overcome low sensitivity problems of the 13C NMR method, we have used a high-power probe of 20 mm o.d. sample tube specially designed for the measurements at low concentrations. To verify the generality of the method, first we should establish reliable NMR criteria for the determination of protein (peptide) location in membranes. We examine whether the location and orientation of peptides in membranes can be reproduced or not by the 13C NMR method, by using two peptides with contrasting orientations in membranes. One is a pentadecapeptide gramicidin A (GA) which forms a transmembrane channel with deep penetration into the hydrophobic bilayer core.9 The other is a synthetic peptide Ac-18A-NH2 which has been designed for lipid-associated structural motif of apoA-I in plasma.10-13 According to a breakthrough in binding site determination by the recent X-ray diffraction study,5 Ac-18A-NH2 is bound superficially to multilamellar phospholipid bilayers with the helix axis oriented parallel to the bilayer interface. To show how reliable the NMR method is to complement the X-ray method, the Ac-18A-NH2 binding site in membranes is to be specified independently by NMR. Here we focus on the binding site of GA and Ac-18A-NH2 in unilamellar vesicles of egg yolk phosphatidylcholine (EPC). We show that the 13C NMR information can reproduce not only the deep penetration of GA in bilayers but also the superficial binding of Ac-18A-NH2 reported by the X-ray diffraction method.4,5 Next we apply our method to the binding site determination of proteins in membranes, using native apoA-I in plasma that is bound to lipid bilayers and emulsions. ApoA-I, the major protein constitutent of high-density lipoproteins (HDL), is the 243 amino acid polypeptide consisting of 10 putative tandem amphipathic R-helices.10,11,14,15 The binding of apoA-I to lipid particle surfaces plays a key role in controlling the HDL stability, lipid transport, and metabolism.10,14-16 To understand these functions involving physiological formation of lipoprotein particles, the structure and mechanism of apoA-I/lipid binding in such particles are crucial subjects to be clarified. One of the most important characteristics of apoA-I/lipid binding is its exchangeability. The exchangeable binding can be achieved only by the superficial association with lipid particles, because any deep insertion of apoA-I into the inner particle core prohibits the dissociation necessary for the biological functions. We focus on how deep apoA-I is located in lipoprotein particles. Since no direct information about apoA-I location and depth has been available yet, it is of great importance to make an extensive application of the NMR method to the protein binding. In this study, the apoA-I binding in EPC small unilamellar vesicles (SUV) is studied because the stability of small-sized lipoproteins such as HDL is due to the apoA-I binding, which is in favor of the highly curved surfaces.16,17 To confirm the curvature dependence, the apoA-I binding in SUV is compared with that in large unilamellar vesicles (LUV). The binding site of apoA-I is also compared with Ac-18A-NH2 binding in SUV and LUV, to examine whether Ac-18A-NH2 is a good model for amphipathic helices of native apoA-I. ApoA-I is accommodated in triglyceride-rich large emulsion-like lipoproteins, chylomicrons (80-1000 nm in diameter), where the protein is assumed to intercalate between the polar surface phospholipids.11 Thus we finally extend our method to the binding site deter-

J. Phys. Chem. B, Vol. 105, No. 50, 2001 12617 mination of apoA-I in triolein (TO)-EPC emulsions, as a proteinfree model of chylomicrons in plasma. Materials and Methods Materials. GA (Dubos) was obtained from Sigma. Ac-18ANH2 (MW 2242.5) was purchased from Takara Shuzo and used without further purification. The purity was 98.0% from RPHPLC. ApoA-I was isolated from pig plasma. Detailed procedures of the isolation were described elsewhere.18,19 The purity was found to be 97.5% from the HPLC analysis. EPC was kindly provided by Asahi Kasei Co. The purity determined by thin-layer chromatography was over 99.5%. TO was purchased from Sigma. Heavy water (D2O, 99.9% D) was used as received from Aldrich. All other chemicals were of special grade from Wako Pure Chemical Industies, Ltd. Sample Preparation. SUV of EPC were prepared by ultrasonic irradiation followed by an extrusion using Nucleopore filters with 50 nm pore diameter. LUV were obtained by an extrusion method using 100 nm pore size filter. The average diameters of SUV and LUV determined from quasielastic light scattering were 30 and 100 nm, respectively. TO-EPC emulsions with 100 nm diameter were prepared as described20,21 using a high-pressure emulsifier (Nanomizer, Nanomizer Inc., Tokyo). The molar ratio of TO/EPC was 85:15. Phosphate-buffered saline (PBS) containing 140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, and 1 mM KH2PO4 in D2O was used to adjust the solution pH at 7.4. SUV containing 10 mol % GA was prepared by mixing the chloroform solutions of EPC and GA followed by the solvent evaporation and ultrasonication in D2O. For the binding of Ac18A-NH2 or apoA-I to vesicles and emulsions, a desired amount of Ac-18A-NH2 or apoA-I in PBS was mixed with lipid dispersions prior to the NMR measurement. Molar ratios of the Ac-18A-NH2 peptide to the outer EPC layer of the vesicles were fixed to 1:20 by preparing 0.3 mM Ac-18A-NH2/9 mM SUV and 0.5 mM Ac-18A-NH2/20 mM LUV. The final concentrations of the apoA-I/EPC were 8 µM apoA-I/6 mM SUV, 9 µM apoA-I/50 mM LUV, and 10 µM apoA-I/7 mM emulsions. The mixing ratios guarantee the sufficient apoA-I binding; more than 90% of the maximum binding amount to the bilayer and emulsion surfaces. Neither vesicle fusion, aggregation, nor disruption of the membrane structure was detected according to the NMR analysis of the EPC signals. NMR Spectroscopy. 13C NMR spectra were recorded at a frequency of 67.7 MHz without sample rotation, using a highresolution, multinuclear, and multipurpose NMR spectrometer (JEOL JNM-EX270 wide-bore type) equipped with an Oxford superconductor magnet (6.35 T). A high-power probe of 20 mm o.d. sample tube was specially designed for the natural abundance 13C NMR measurements at low concentrations. Free induction decays (FID) were accumulated 30 000-170 000 times. Line broadening of 3 Hz was applied before Fourier transformation. All measurements were performed at 37.0 ( 0.5 °C. 2,2-Dimethyl-2-silapentane-5-sulfonate (DSS), contained in a capillary, was used as an external reference. Chemical shifts were accurate within (0.02 ppm. Results and Discussion Binding Site of GA in Membranes. A transmembrane channel peptide GA is oriented parallel to the lipid alkyl chains of the bilayer with deep penetration into the hydrophobic interior.9 Hence we expect that the NMR data indicate strong interactions of GA with the lipid chain core. GA binding is an appropriate model to examine whether the NMR data reproduce the deep penetration in membranes. Here the 13C NMR sinals

12618 J. Phys. Chem. B, Vol. 105, No. 50, 2001

Figure 1. Chemical shift change of each 13C NMR signal of EPC vesicles and emulsions induced by peptides and proteins in systems (a) GA+SUV together with the image of GA transmembrane orientation, (b) Ac-18A-NH2+LUV, (c) Ac-18A-NH2+SUV, (d) apoAI+SUV, (e) apoA-I+LUV, and (f) apoA-I+TO/PC emulsions. Negative values mean an upfield shift. The polar headgroup and the interfacial and hydrophobic parts of the membranes are displayed in green, orange, and red colors, respectively. The X-ray diffraction result of the distribution of Ac-18A-NH2 helix in dioleoyl-PC bilayers9 is superimposed as a solid line b′.

of GA are almost unresolved due to the static and dynamic broadening as a result of the anisotropic structure and restricted motions of rigid GA helices. No appearance of GA signals makes it possible to evaluate the peptide binding site in membranes from lipid NMR signals. First we compare the chemical shifts of the respective13C NMR signals of EPC before and after the peptide binding. Here we assume the empirical rule that NMR signals shift to a lower field when molecules are hydrated in a polar environment.22,23 NMR chemical shifts are used as a sensitive indicator to probe the location in membranes.7,8 In Figure 1a, the membrane perturbation by GA is shown as the chemical shift change of each carbon atom site of EPC SUV. As expected, most significant chemical shift changes are observed at the EPC methylene chains (CH2)n in the hydrophobic core as well as the carbonyl at the bilayer interface. The result shows that GA is penetrated deeply into the hydrophobic core of the lipid bilayer. The transmembrane location of GA is also confirmed by the line width analysis24 of the membrane 13C NMR signals shown in Table 1. The line width broadening is most characteristic at the lipid chain methylene and methyl sites (CH2)n, (ω-2)CH2, (ω-1)CH2, and ω-CH3 in the bilayer core, although the signals

Okamura et al. are moderately broadened at the other carbon atom sites in the bilayer headgroup and the interfacial region.25 All results are consistent with the deep penetration of GA into the hydrophobic core of the bilayer; our NMR method actually reproduces the transmembrane binding picture of GA on the molecular level. Binding Site of Ac-18A-NH2 in Membranes. To examine whether the NMR method is compatible with the superficial binding of Ac-18A-NH2 in oriented bilayers, we have determined the Ac-18A-NH2 binding site in EPC LUV and SUV with low and high surface curvatures, respectively, in comparison with the X-ray diffraction data.5 We have demonstrated that 13C NMR and X-ray results of Ac-18A-NH have the identical 2 evidence for the binding site in the bilayer. Here almost no overlapping is shown by Ac-18A-NH2 and EPC NMR signals, as in the case of GA. LUV System. Figure 1b shows that a downfield chemical shift change is induced by Ac-18A-NH2 at the lipid glycerol C3 site. The peptide binding induces a marked line width broadening at the glycerol C2 and carbonyl sites, as seen in Table 1. All results indicate that Ac-18A-NH2 is bound preferentially to the glycerol and carbonyl sites at the LUV interfaces. It is to be noted that our NMR findings agree well with the site preference of Ac-18A-NH2 binding in oriented bilayers by the X-ray diffraction method.5 Assuming Gaussian transbilayer distribution of the Ac-18A-NH2 helix, the X-ray result has demonstrated that the helix center of Ac-18A-NH2 is located close to the lipid glycerol group at the bilayer interface.5 Our NMR data follow the distribution of Ac-18A-NH2 helix in bilayers,5 which is superimposed as curve b′ in Figure 1b. The agreement of the NMR and X-ray results is not only crucial for the determination of the peptide binding site in bilayers but also shows how reliable the NMR method is in the determination of the peptide binding site in membranes. Here we have ascertained the NMR empirical rule1,2 that a downfield chemical shift means hydration. SUV System. Membrane pertubation by the Ac-18A-NH2 binding in SUV is different from that in LUV mentioned above; compare Figure 1c with Figure 1b. Most significant is the upfield chemical shift at the EPC carbonyl group in the intermediate polarity zone of SUV. The upfield chemical shift means dehydration according to the empirical rule stated above. The carbonyl group is most susceptible to the hydration state of lipid bilayers. In the liquid crystalline phase, the carbonyl is the most inner site into which water can penetrate.26-28 Thus we can say that Ac-18A-NH2 penetrates into the bilayer interfacial zone, competing with the penetrating water, and excluding the water molecules from the carbonyl site at the SUV interface. All results demonstrate a high affinity of Ac-18A-NH2 for the ester carbonyl site of SUV. On the other hand, the polar headgroup and the hydrophobic chains are much less perturbed. Relatively small changes are shown in the chemical shift of the choline N(CH3)3, CH2N, and CH2OP and the alkyl chain. The results indicate that Ac-18A-NH2 weakly interacts with bilayer headgroup and the bilayer core. The difference of Ac-18A-NH2 bindings in SUV and LUV is also confirmed by the line width analysis in Table 1. In SUV, the line broadening effect is most protruding at the EPC glycerol C2, C1, and carbonyl sites. No significant line width increases are shown by the hydrophilic choline group and the inner sites of the alkyl chains. Large surface curvatures and hydration of the SUV interfaces allow the enhanced motional freedom of the carbonyl group; its large fluctuation can be repressed (energetically stabilized) by the Ac-18A-NH2 binding. Finally the NMR evidence for an Ac-18A-NH2 binding site in highly

Determination of Peptide and Protein Binding Sites

J. Phys. Chem. B, Vol. 105, No. 50, 2001 12619

TABLE 1: Line Width Broadening of the 13C NMR Signals of EPC SUV and LUV in the Presence of GA, Ac-18A-NH2, and apoA-I halfwidth/ Hza carbon atom site

chemical shift/ppmb

SUV only

+GA

N(CH3)3 CH2N CH2OP glycerol C3c glycerol C2c glycerol C1c carbonyle R-CH2 β-CH2 CHdCHCH2CH2 dCHCH2CHd (CH2)n (ω-2)CH2 (ω-1)CH2 ω-CH3

56.69 68.67 62.04 66.28 73.20 65.65 176.21 36.63 27.53 29.70 28.10 32.33 34.49 25.17 16.39

11.2 16.8 12.9 14.3 32.8 17.2 15.1 20.2 17.4 12.1 10.6 65.0 12.9 14.6 8.4

24.0 39.0 24.0 29.0 ∼80 ∼27 40.0 39.0 n.d.d 28.5 32.4 96.5 22.0 33.5 37.4

+Ac-18A-NH2 +apoA-I 16.2 24.4 16.3 24.1 n.d.d ∼40 29.1 28.6 ∼20 24.1 12.9 66.6 12.3 13.4 11.2

14.6 21.0 15.1 15.1 125.4 30.8 23.5 23.5 17.4 16.0 12.3 68.9 13.4 14.0 9.5

LUV only +Ac-18A-NH2 +apoA-I 14.0 24.6 21.3 25.2 107.5 n.r.f 26.9 30.0 25.8 28.0 16.8 71.1 14.6 16.8 11.2

18.5 24.6 21.8 28.6 n.d.d n.r.f 45.4 26.9 24.1 25.2 17.4 71.1 15.7 17.9 13.4

12.9 22.4 21.3 23.0 n.r.f n.r.f 28.0 34.7 28.6 29.7 18.5 71.7 15.1 16.8 11.2

a With an experimental error, (0.6 Hz. b Chemical shift values of EPC SUV relative to DSS. c Glycerol C3, the glycerol CH2 next to the phosphate group; C2, the glycerol CH; C1, the glycerol CH2 next to the ester carbonyl group. d Not determined because of the signal broadening. e Value for the signal assigned to the outer leaflet of the bilayer. f Not resolved.

curved SUV interfaces is valuable because no direct information has been available for the peptide binding in spherical lipid particles. Binding Site of apoA-I in Membranes. In the previous section, we have shown how reliable the NMR method is in the determination of the peptide binding site in membranes. We have demonstrated the applicability of the natural abundance 13C NMR method to the binding site determination of the two peptides with different orientations. Here we apply the method to the determination of native apoA-I binding site in membranes; no direct evidence has been provided yet. If Ac-18A-NH2 is a good model for the native apoA-I in plasma, apoA-I is expected to bind superficially to the membrane surfaces. We investigate whether the apoA-I binding site in membranes is consistent with that of the model peptide Ac-18A-NH2 mentioned above. In a “snorkel” model,11,13 a deep penetration of the amphipathic helices into the hydrophobic interior of the lipid is assumed, with the wedge-shaped configuration of the helices assisted by the relatively hydrophobic side chain of the lysine residues. We should examine whether this model is compatible or not with the NMR information obtained in this work. We also report how apoA-I is bound to lipid emulsions, motivated by the biological importance of the system where plasma lipoproteins have an emulsion structure consisting of a hydrophobic core of cholesteryl esters and triglycerides, surrounded by a surface monolayer of phospholipids, unesterified cholesterol, and apolipoproteins.10,15 13C NMR Spectra of the apoA-I/SUV System. If native apoA-I and its model peptide Ac-18A-NH2 have a common binding site, we expect that apoA-I binding induces significant changes in the SUV interface. In fact, the comparison of the 13C NMR spectra of SUV in Figures 2a and b shows that apoA-I binding induces drastic changes at the lipid ester carbonyl site at 176 ppm and the glycerol CH (C2) site at 73 ppm. An important change is the merging of the doublet of the ester carbonyl signal that is assigned to the inner and outer leaflets of the bilayer; see the inset of Figure 2. Another change is the extreme line width broadening of the glycerol C2 signal as represented by the dotted line. The signal broadening is also recognized by the halfwidth increase of the glycerol C2 from 33 to 125 Hz in Table 1. The spectral changes at the glycerol and carbonyl sites suggest that the binding site of apoA-I involves these two sites in the intermediate polarity zone at the SUV interface. The line broadening is due to the restriction in the segmental motions

Figure 2. Natural abundance 13C NMR spectra of (a) EPC SUV and (b) apoA-I/EPC SUV system. The glycerol CH site is designated by the arrow. The carbonyl region of the 13C NMR spectra is expanded on the inset. Asterisks denote the reference signals of DSS.

of glycerol C2 group by the apoA-I binding; the line width reflects the atomic and segmental motions.1 Details of the line broadening effect of apoA-I binding will be discussed later in relevance to the dynamic properties of membranes. To obtain the information about apoA-I in lipid-free and lipidbound states, we have attempted to compare the 13C NMR spectra of apoA-I in the presence and absence of SUV. ApoA-I signals were, however, unresolved as in the case of GA. This is due to the static and dynamic broadening as a result of the anisotropic structure and restriction in the motional freedom of apoA-I molecules. The restricted motions are closely related to the rigid structure of apoA-I with an R-helical content of ∼70%.29 No appearance of the apoA-I signals, fortunately, makes it possible to evaluate the protein binding site in

12620 J. Phys. Chem. B, Vol. 105, No. 50, 2001 membranes from lipid NMR signals, as described in the former two peptides. Chemical Shift Analysis. To specify apoA-I binding site in membranes, we compare the chemical shifts of the respective13C NMR signals of EPC. In Figure 1d is shown membrane perturbation by apoA-I. The most significant is the upfield chemical shift of the ester carbonyl group. The upfield chemical shift means dehydration. The specific effect of apoA-I on the lipid carbonyl group is also noticed by the signal merging shown in the inset of Figure 2. Thus we can say that apoA-I penetrates into the bilayer interfacial zone and excludes the water molecules from the carbonyl site at the SUV interface, just as the Ac18A-NH2 binding. All results demonstrate a high affinity of apoA-I for the ester carbonyl group, the central part of the apoA-I binding in membranes. The polar headgroup and the hydrophobic chains are much less perturbed by apoA-I. As can be seen in Figure 1d, there are relatively small changes in the chemical shift at the choline N(CH3)3, CH2N, and CH2OP and the alkyl chain. The results indicate that apoA-I weakly interacts with the bilayer headgroup and the bilayer core. This is consistent with the line width analysis in Table 1 as described later in detail. The comparison of Figures 1c and d shows that membrane perturbation of apoA-I and its model peptide Ac-18A-NH2 is quite similar. We can regard Ac-18A-NH2 binding in SUV as a good model of apoA-I binding at the highly curved bilayer interfaces. The marked differences in the binding pattern between GA and apoA-I (Figures 1a and d) deny the deep transmembrane location of apoA-I helices in membranes. This shows that the helical axes of apoA-I are oriented not perpendicular but parallel to the bilayer interface. The rather mild binding feature of apoA-I is in reasonable agreement with the exchangeability of apolipoprotein bindings in plasma lipoproteins. It is consistent with the X-ray diffraction study concluding that modest bilayer perturbation is a general feature of amphipathic helix bindings at low concentrations.6 Finally the strong affinity of apoA-I for the highly curved bilayer interfaces is confirmed by comparing the membrane perturbation in SUV and LUV; see Figures 1d and e. In LUV, changes in the chemical shifts are small just as expected. It is understandable in view of one-order lower protein binding affinity to LUV as compared with SUV.30 It is consistent with the negligibly small line broadening effect of apoA-I on the LUV signals summarized in Table 1. Line Width Analysis. ApoA-I binding is expected to alter the membrane dynamics, especially near the binding site. Table 1 summarizes the halfwidth changes of the 13C NMR signal of EPC SUV and LUV induced by the apoA-I binding. In SUV, the line broadening effect by apoA-I is most remarkable at the glycerol C2 site. The signal broadening is also recognized at the glycerol C1 and ester carbonyl sites. The result means the special affinity of apoA-I to the lipid glycerol and carbonyl sites of SUV; their motions are reducecd by the protein binding. It is concluded that apoA-I binds preferably to the glycerol C2 and C1 sites as well as the carbonyl at the SUV interface. It is interesting that the site preference of apoA-I is similar to the case of the model peptide in oriented lipid bilayers.5 The NMR signal broadening is not discernible at the EPC headgroup of SUV. As shown in Table 1, apoA-I induces only small line width increase from 11 to 15 Hz at the choline methyl group. The situation is the same at the two choline methylenes CH2N and CH2OP, the chain CHdCHCH2CH2 and dCHCH2CHd. In contrast, the segmental motions of the most inner sites of the CH chains are unaffected by apoA-I; no significant line

Okamura et al. broadening is induced for the carbon atom sites (ω-2)CH2, (ω1)CH2, and ω-CH3. Thus apoA-I binding induces small structural perturbation of the SUV headgroup and no significant perturbation of the inner bilayer core. In LUV no significant line width changes are induced by apoA-I; see Table 1. This indicates that apoA-I does not alter the dynamic properties of membranes with low surface curvatures. It is consistent with the preferable binding of apoA-I to highly curved bilayer interfaces. ApoA-I Binding to Emulsions. To confirm the generality of apoA-I binding site in membranes, we finally apply the 13C NMR method to apoA-I binding to a protein-free model of plasma lipoproteins, 100 nm-sized emulsions of TO core and a surface EPC monolayer. The superficial binding is disclosed; the chemical shift in Figure 1f indicates significant upfield shifts at the EPC glycerol C3 and carbonyl sites of the emulsion surface monolayer. This indicates dehydration at the emulsion surfaces due to the binding of apoA-I. The picture is analogous to the apoA-I binding at the SUV interfaces. The apoA-I binding affinity to emulsion surfaces is similar to SUV rather than LUV. This is due to the differences in the dynamic structure between emulsion and LUV surfaces; the ester carbonyl site of the emulsion surface monolayer is proved to be highly fluctuating as seen in the SUV interfaces.30 Loose coupling or motional freedom of the lipid segment at SUV and emulsion surfaces is preferred to the apoA-I binding. Thus it is concluded that the large segmental fluctuation at lipid particle interfaces is closely related to the binding site of apoA-I. The location of apoA-I at the emulsion surfaces is consistent with the fluorescence studies suggesting superficial binding of apoA-I in cholesterol-enriched EPC bilayers31 and chicken HDL particles.32 Conclusions In the present work, we propose the natural abundance 13C NMR method as a simple but general method for the reliable determination of the location and depth of proteins and peptides in membranes without 13C enrichment. To establish reliable NMR criteria for the determination of protein (peptide) location in membranes, we examine whether the location and orientation of peptides in membranes can be reproduced or not by the 13C NMR method, by using two peptides GA and Ac-18A-NH2 with contrasting orientations in membranes. We have first demonstrated that both NMR and X-ray diffraction results have the identical atomic-level evidence for the superficial binding of Ac-18A-NH2 and how reliable the empirical NMR chemical shift rule is in the binding site determination. The method was then applied to determine the binding site of native apoA-I in lipid bilayers and emulsions; no direct evidence has been provided yet. The shallow penetration of apoA-I was found in SUV and TO-EPC emulsions; apoA-I modulates the degree of hydration and segmental motions at the lipid membrane interface. The key binding site of apoA-I is the intermediate polarity region of the membranes including lipid glycerol and ester carbonyl group. This is in contrast to the deep penetration of GA into the hydrophobic membrane core. The shallow penetration is relevant to the exchangeability of apoA-I in the process of lipoprotein metabolism; the superficial binding at the lipid particle interfaces is favorable for the association and dissociation of apoA-I in lipoprotein particles. Acknowledgment. This work was supported by the Grantin-Aid for Creative Scientific Research (13NP0201) “Collaboratory on Electron CorrelationssToward a New Research Network between Physics and Chemistry” from the Ministry

Determination of Peptide and Protein Binding Sites of Education , Culture, Sports, Science and Technology and by the Grant-in-Aid for Scientific Research (13440179) from JSPS. References and Notes (1) Okamura, E.; Nakahara, M. Liquid Interfaces in Chemical, Biological, and Pharmaceutical Applications; Volkov, A. G., Ed.; Marcel Dekker: New York, 2001; pp 775-805. (2) Okamura, E.; Nakahara, M. J. Phys. Chem. B 1999, 103, 3505. (3) Okamura, E.; Kakitsubo, R.; Nakahara, M. Langmuir 1999, 15, 8332. (4) White, S. H.; Wimley, W. C. Biochim. Biophys. Acta 1998, 1376, 339. (5) Hristova, K.; Wimley, W. C.; Mishra, V. K.; Anantharamiah, G. M.; Segrest, J. P.; White, S. H. J. Mol. Biol. 1999, 290, 99. (6) Hristova, K.; Dempsey, C. E.; White, S. H. Biophys. J. 2001, 80, 801. (7) Prosser, R. S.; Luchette, P. A.; Westerman, P. W. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9967. (8) Prosser, R. S.; Luchette, P. A.; Westerman, P. W.; Rozek, A.; Hancock, R. E. W. Biophys. J. 2001, 80, 1416. (9) Killian, J. A. Biochim. Biophys. Acta 1992, 1113, 391 and references therein. (10) Brouillette, C. G.; Anantharamaiah, G. M. Biochim. Biophys. Acta 1995, 1265, 103. (11) Segrest, J. P.; Garber, D. W.; Brouillette, C. G.; Harvey, S. C.; Anantharamaiah, G. M. AdV. Protein Chem. 1994, 45, 303. (12) Segrest, J. P.; De Loof, H.; Dohlman, J. G.; Brouillette, C. G.; Anantharamaiah, G. M. Proteins 1990, 8, 103. (13) Segrest, J. P.; Jones, M. K.; De Loof, H.; Brouillette, C. G.; Venkatachalapathi, Y. V.; Anantharamaiah, G. M. J. Lipid Res. 1992, 33, 141. (14) Narayanaswami, V.; Ryan, R. O. Biochim. Biophys. Acta. 2000, 1483, 15. (15) Brouillette, C. G.; Anantharamaiah, G. M.; Engler, J. A.; Borhani, D. W. Biochim. Biophys. Acta 2001, 1531, 4. (16) Small, D. M. Plasma Lipoproteins and Coronary Artery Disease; Kreisberg, R. A.; Segrest, J. P., Eds.; Blackwell Scientific Publications Inc.: London, 1992; pp 57-91.

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