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Two Homologous Apolipoprotein AI Mimetic Peptides

Two Homologous Apolipoprotein AI Mimetic Peptides THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 49, Issue of December 3, pp. 51404–51414, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. RELATIONSHIP BETWEEN MEMBRANE INTERACTIONS AND BIOLOGICAL ACTIVITY* Received for publication, July 28, 2004, and in revised form, September 8, 2004 Published, JBC Papers in Press, September 8, 2004, DOI 10.1074/jbc.M408581200 Richard M. Epand‡§, Raquel F. Epand‡, Brian G. Sayer‡, Geeta Datta¶, Manjula Chaddha¶, and G. M. Anantharamaiah¶ From the ‡Departments of Biochemistry and Biomedical Sciences and Chemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada and the ¶Departments of Medicine, Biochemistry, and Molecular Genetics and the Atherosclerosis Research Unit, University of Alabama at Birmingham, Birmingham, Alabama 35294 Two related 18-amino acid, class A, amphipathic heli- density lipoprotein-like particles containing peptide, choles- cal peptides termed 3F-2 and 3F were chosen for this terol, apo A-I and paraoxonase, an enzyme capable of convert- study. Although they have identical amino acid compo- ing pro-inflammatory high density lipoprotein into anti-inflam- sitions and many similar biophysical properties, 3F-2 is matory high density lipoprotein (4). The peptide 4F has also more potent than 3F as an apolipoprotein AI mimetic been tested in vitro and shown to effectively inhibit lytic pep- peptide. The two peptides exhibit similar gross confor- tide-induced hemolysis, inhibit oxidized phospholipid-induced mational properties, forming structures of high helical monocyte chemotaxis, scavenge lipid hydroperoxides from LDL content on a membrane surface. However, the thermal (5), and maintain endothelial nitric-oxide synthetase activity in denaturation transition of 3F-2 is more cooperative, sug- the presence of atherogenic concentrations of LDL (6). To study gesting a higher degree of oligomerization on the mem- the relationship of peptide structure to anti-atherosclerogenic brane. Both 3F-2 and 3F promote the segregation of potency, we studied the properties of four related 18-amino cholesterol in membranes containing phosphatidylcho- acid, class A amphipathic helical peptides (5). All of these line and cholesterol, but 3F-2 exhibits a greater selectiv- peptides had identical amino acid compositions and very sim- ity for partitioning into cholesterol-depleted regions of ilar physical properties, yet two of these peptides, 3F-1 and the membrane. Magic angle spinning/NMR studies indi- 3F-2, were more potent in inhibiting lytic peptide-induced he- cate that the aromatic residues of 3F-2 are stacked in the molysis, inhibiting oxidized phospholipid-induced monocyte presence of lipid. The aromatic side chains of this pep- chemotaxis, and scavenging lipid hydroperoxides from LDL com- tide also penetrate more deeply into membranes of 3 14 pared with the analogs 3F and 3F (5). In the present work we phosphatidylcholine with cholesterol compared with compare the interaction with phospholipid bilayers with and 3F . Using the fluorescent probe, 1,3-dipyrenylpropane, we monitored the properties of the lipid hydrocarbon without cholesterol, of the most potent peptide of this series, 3F-2 environment. 3F-2 had a greater effect in altering the (Ac-DKWKAVYDKFAEAFKEFL-NH ), and the least potent properties of the hydrocarbon region of the membrane. among these peptides, 3F (Ac-DWLKAFYDKVAEKFKEAF- The results are consistent with our proposed model of NH ) (Fig. 1). We have previously shown that the potent analog the effect of peptide shape on the nature of the differ- 4F that has four, rather than three Phe, is capable of forming ence in peptide insertion into the bilayer. cholesterol-rich domains by preferentially interacting with re- gions of the membrane that are depleted of cholesterol (7). 3F-2 exhibits similar biological potency to 4F but has somewhat less There is growing evidence that certain apo A-I mimetic, activity in inhibiting oxidized phospholipid-induced monocyte class A amphipathic helical peptides can be used to inhibit chemotaxis, about the same activity in scavenging lipid hy- atherosclerosis (1). The oral administration of peptide 4F syn- droperoxides from LDL and greater activity in inhibiting lytic thesized from all-D amino acids (D-4F) protects mice from peptide-induced hemolysis (5). In contrast, although 3F is a diet-induced atherosclerosis without altering plasma choles- class A amphipathic helical peptide with some anti-atherosclero- terol levels (2, 3). Preliminary studies also suggest that oral genic activity, it has a much weaker potency than either 3F-2 or administration of D-4F to LDL receptor null and apo E null 4F in the activities mentioned. This difference in potency is mice causes the rapid formation and clearance of small high reflected in differences in the red edge effect in Trp emission from the peptide and in the quenching of 2-(3 (diphenylhexatrienyl)- propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (5). In * This work was supported by Grant MT-7654 from the Canadian this study we further evaluate the interaction of 3F-2 and 3F Institutes of Health Research and Grants HL 34343 and RO1 HL 65663 with model membranes using NMR, DSC, CD, and fluorescence. from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance MATERIALS AND METHODS with 18 U.S.C. Section 1734 solely to indicate this fact. Lipids—The lipids used in this study were obtained from Avanti § To whom correspondence should be addressed. E-mail: epand@ Polar Lipids (Alabaster, AL). The purity of the phospholipids was ver- mcmaster.ca. 1 ified by measuring the cooperativity and temperature of the phase The abbreviations used are: apo, plasma apolipoprotein; LDL, low transition using DSC. density lipoprotein; PC, phosphatidylcholine; PO, 1-palmitoyl-2-oleoyl; Peptide Synthesis—The peptides were synthesized by the solid phase SO, 1-stearoyl-2-oleoyl; PC P, 1,3-dipyrenylpropane; MAS, magic angle method with a Protein Technologies PS-3 automatic peptide synthe- spinning; NOESY, nuclear Overhauser enhancement spectroscopy; sizer using the procedures described previously (2, 8). The peptides LUV, large unilamellar vesicle; DSC, differential scanning calorimetry; were purified using a preparative HPLC system (Beckman Gold), and HPLC, high pressure liquid chromatography; PIPES, 1,4-piperazinedi- ethanesulfonic acid; I , intensity of excimer emission; I , intensity of the purity of the peptides was ascertained by mass spectral analysis e m monomer emission. and analytical HPLC. 51404 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Membrane Interactions of 3F Analogs 51405 FIG.1. Helical wheel representa- tion of 3F-2 and 3F and molecular models of these peptides with sur- rounding phosphatidylcholine. The wheel is projected along the axis of the helix from the N to the C terminus with the hydrophobic side facing downward. The primary structure is given above each wheel diagram. The amino acid composi- tion of both peptides is the same. The sequence is different. The plus and minus signs denote the charges on the amino acids at neutral pH. The red color denotes an acidic residue, the blue color denotes a basic residue, and the bold black denotes aromatic residues. Concentrations of Peptide and Lipid—The concentrations of peptide Temperature heating or cooling scans were performed at a rate of solutions in buffer were determined spectrophotometrically using the 2 °C/min, with a 30-s equilibration pause at each point for thermal 1 1 absorbance at 280 nm and an extinction coefficient of 6970 cm M , equilibration. Ellipticity at 222 nm was measured as a function of calculated from the Tyr and Trp content. Phospholipid concentration temperature between 25 and 95 °C and then cooling back to 25 °C to was determined by phosphate analysis (9). assess the reversibility of denaturation. Preparation of Samples for DSC and NMR Experiments—Phospho- Large Unilamellar Vesicles (LUVs)—Lipid films were made by dis- lipid and cholesterol were dissolved in chloroform/methanol (2/1, v/v). solving appropriate amounts of lipid in a mixture of chloroform/meth- For samples containing peptide, an aliquot of a solution of the peptide anol (2/1, v/v) and dried in a test tube under nitrogen to deposit the lipid in methanol was added to the lipid solution in chloroform/methanol. as a thin film on the wall of the tube. Final traces of solvent were The solvent was then evaporated under a stream of nitrogen with removed in a vacuum chamber attached to a liquid nitrogen trap for 2–3 constant rotation of a test tube so as to deposit a uniform film of lipid h. Dried films were kept under argon gas at 30 °C if not used imme- over the bottom third of the tube. The last traces of solvent were diately. The films were hydrated with buffer, vortexed extensively at removed by placing the tube under high vacuum for at least 2 h. The room temperature, and then subjected to five cycles of freezing and lipid film was then hydrated with 20 mM PIPES, 1 mM EDTA, 150 mM thawing. The homogeneous lipid suspensions were then further pro- NaCl with 0.002% NaN , pH 7.40, and suspended by intermittent cessed by 10 passes through two stacked 0.1-m polycarbonate filters vortexing and heating to 50 °C for 2 min under argon. The samples used (Nucleopore Filtration Products, Pleasanton, CA) in a barrel extruder for NMR analysis were hydrated with the same buffer made in H O (Lipex Biomembranes, Vancouver, Canada), at room temperature. adjusted to a pH meter reading of 7.0 (pD  7.4). The samples used for LUVs were kept on ice and used within a few hours of preparation. NMR were incubated 24 h at 4 °C to allow conversion of anhydrous Tryptophan Fluorescence—Fluorescence emission spectra of the pep- cholesterol crystals to the monohydrate form. For the NMR measure- tides in the presence and absence of LUVs were measured at 25 °C ments, the samples were spun in an Eppendorf centrifuge at room using an SLM Aminco Series II luminescence spectrometer. The exci- temperature. The resulting hydrated pellet was transferred to a 12-l tation wavelength was varied between 280 and 310 nm. The emission capacity Kel-F spherical insert of an 18  4-mm ZrO rotor, attempting 2 scans were recorded and then corrected for inner filter and instrumen- to pack the maximal amount of lipid into the rotor while maintaining tal effects. it wet. Excimer Formation in the Fluorescent Probe PC P—The physical Differential Scanning Calorimetry—Measurements were made im- properties of the hydrophobic region of the membrane and the effects of mediately after sample preparation using a Nano Differential Scanning the 3F peptides were monitored by measuring the fluorescent emission Calorimeter (Calorimetry Sciences Corporation, American Fork, UT). of PC P (Molecular Probes, Eugene, OR). It has been shown that the The scan rate was 2 °C/min, and there was a delay of 5 min between ratio of the intensities of excimer and monomer emission is sensitive to sequential scans in a series to allow for thermal equilibration. DSC the “fluidity” of the membrane interior (12). LUVs were prepared con- curves were analyzed by using the fitting program, DA-2, provided by taining lipids and PC P at a 100:1 molar ratio in Hepes buffer (10 mM Microcal Inc. (Northampton, MA) and plotted with Origin, version 5.0. Hepes, 0.14 M NaCl, 1 mM EDTA, pH 7.4). Florescence measurements Centrifugation Assay for Membrane Binding of 3F Peptides—The were made in 1  1-cm-square quartz cuvettes containing 2 ml of Hepes fraction of peptide bound to the lipid after three heating and cooling buffer at room temperature. The excitation wavelength was 344 nm cycles between 0 and 100 °C was determined by separating the free using slits with 4-nm band pass in both emission and excitation. Emis- peptide by centrifugation. The vesicles with bound protein were pel- sion scans were made between 360 and 550 nm before and after the leted at 200,000  g for 90 min at 25 °C. A clear supernatant was addition of peptide. The results are summarized as the ratio of the separated from the solid pellet and assayed for protein by absorption at intensity of excimer emission at 476 nm to that of monomer emission at 278 nm. The absorption of a blank was subtracted, and a base line, set 389 nm (I /I ). The experiments were repeated twice with two inde- e m at 350 nm for each sample, was used to correct for residual scattering. pendent preparations. The error in I /I for duplicate measurements e m In addition, the amount of lipid in both pellet and supernatant fractions with the same preparations was between 1 and 2%, but between inde- was determined by phosphate analysis. Cholesterol concentration was pendently prepared samples the absolute value of the ratios varied determined by a modified version of the procedure of Zlatkis et al. (10). between 5 and 10%; however, they always exhibited similar relative Briefly, after extraction with chloroform, the organic phase was dried values with and without either of the two peptides. under nitrogen and then placed under vacuum to completely remove the 1 H NOESY MAS NMR—High resolution MAS spectra were acquired solvent. To the dried samples or to the dry cholesterol standards, glacial using a spinning rate of 4 kHz in a Bruker DRX 500 NMR spectrometer. acetic acid was added, followed by a reagent composed of FeCl and 85% Probe temperature was 24  1 °C. The two-dimensional NOESY spec- H PO in sulfuric acid. The samples were placed in a boiling water bath 3 4 tra were obtained using mixing times of 50 and 300 ms. The resonances for 3 min to develop a purple color. After cooling the absorbance was were assigned based on their close similarity to literature values for read at 550 nm. phosphatidylcholine (13), cholesterol (14), and amino acid residues (15). To test for the presence of lipoprotein particles in the supernatant, it was analyzed by nondenaturing PAGE using the method of Laemmli RESULTS (11). An aliquot of the supernatant containing 3 g of peptide was loaded on a 4–20% gel and run under native conditions for 20 h. The gel DSC—Lipid mixtures of SOPC containing 0, 0.3, 0.4, and 0.5 was then stained with colloidal blue and destained with water. mol fractions cholesterol were analyzed by DSC in the presence Circular Dichroism—CD spectra were recorded on an AVIV model of 0, 5, 10, and 15 mol % 3F-2 or 3F . As examples, we present 215 spectropolarimeter using a quartz cell with a 0.1-cm path length. the results from mixtures containing 0 or 15 mol % of each of The cuvette was placed in a jacketed cell holder maintained at the the peptides (Fig. 2). The DSC curves are presented as the desired temperature with circulating thermostatic fluid. The lipid was excess heat capacity/mol cholesterol as in our earlier paper on solubilized by incubation with the peptide followed by sonication to ensure minimal effects of light scattering. 4F (7). Both peptides clearly promote the separation of choles- 51406 Membrane Interactions of 3F Analogs FIG.2. Differential scanning calorimetry of SOPC:cholesterol mixtures with 0, 5, 10, or 15 mol % 3F-2 or 3F . Scan rate 2 K/min. The rows correspond to lipid compositions of 7:3 (top row), 6:4 (middle row), and 1:1 (bottom row) SOPC:cholesterol, respectively. The columns correspond to lipid alone (first column) or lipid with the addition of 15% of either 3F-2 (second column)or3F (third column). Lipid concentration is 2.5 mg/ml in 20 mM PIPES, 1 mM EDTA, 150 mM NaCl with 0.002% NaN , pH 7.40. Sequential heating and cooling scans between 0 and 100 °C. The numbers are the order in which the scans were carried out, with scans 1 and 3 being heating scans, each of which was followed by one of the cooling scans 2 or 4. Scans were displaced along the y axis for clarity of presentation. TABLE I Enthalpy of the polymorphic transition of anhydrous cholesterol crystals The values are in cal/mol cholesterol determined from areas under the transitions at 36 °C on the first DSC heating scan. 3F-2 3F Cholesterol in SOPC 510 15510 15 30% 00000 85 40% 0 170 55 55 95 160 50% 250 225 400 200 200 225 terol into crystalline domains at higher mol fractions of choles- been found previously, albeit to a lesser extent, with the pep- terol and of peptide (Table I). The cholesterol crystals formed tide 4F (7). Scanning only up to 50 °C can eliminate much of are in a metastable state and disappear after sequential heat- this loss (16), but the higher temperatures are required to ing and cooling scans between 0 and 100 °C (Fig. 2). This has observe the unfolding transition of the peptide. 3F causes Membrane Interactions of 3F Analogs 51407 TABLE II Enthalpy of the chain melting transition of SOPC The values are in cal/mol SOPC determined from the average area under the transitions at 6 °C on the DSC cooling scans. 3F-2 3F Cholesterol in SOPC 0 5 10 15 5 10 15 0% 4000 ND ND 2200 ND ND 2400 30% 500 800 345 278 515 515 530 40% 350 0 0 0 470 570 265 50% 210 0 0 0 300 220 200 ND, not determined. FIG.3. Circular dichroism spectra of 3F-2 (left) and 3F (right)in20mM phosphate buffer, 0.14 M NaCl, 1 mM EDTA, pH 7.4 (A), in the presence of small unilamellar vesicles of 1 mM SOPC with 15% peptide (B), and small unilamellar vesicles of 1 mM SOPC:cholesterol (1:1) with 15% pep- tide (C). The lipid was solubilized by the peptide, but the mixture was also soni- cated to reduce the possibility of scatter- ing artifacts. The spectra were taken in a 1-mm cell at 25 °C. separation of cholesterol crystals at lower mol fractions of cho- is readily observed only with SOPC in the absence of choles- lesterol and peptide than is required with 3F-2. A more marked terol (not shown), but the transition is quite prominent in scans difference is observed in the peak at the lowest transition with 3F-2 (Fig. 2). The transition temperature is slightly lower temperature that is ascribed to the gel to liquid crystalline on cooling than on heating. transition of SOPC. The enthalpy of this transition can be CD—The CD spectra in buffer of both 3F-2 and 3F exhibit reasonably estimated from cooling scans. For pure SOPC (data some dependence on peptide concentration (5). The addition of not shown), the transition occurs at 5.5 °C on cooling at 2 K/ SOPC or SOPC:cholesterol (1:1) to 3F-2 at 100 M peptide min with a transition enthalpy of 4 kcal/mol (16). With pure results in little change in the secondary structure of 3F-2 (Fig. SOPC without cholesterol, the addition of 15 mol % 3F-2 lowers 3) but slightly increases the magnitude of the CD for 3F (Fig. the enthalpy to 2.2 kcal/mol and to 2.4 kcal/mol with 3F (data 3). The temperature dependence of the spectrum shows a sub- not shown). With mixtures of cholesterol and SOPC the addi- stantial loss of secondary structure on heating to 95 °C with tion of increasing concentrations of 3F-2 eliminates this tran- both 3F-2 and with 3F (Fig. 4). The thermal transition is sition, whereas with 3F the enthalpy of this transition is broad with some hysteresis on heating and cooling. slightly increased (Table II). Solubilization of Lipid and Peptide—Mixtures of SOPC, cho- The highest temperature transition observed in the DSC lesterol, and the peptide 3F-2 or 3F were centrifuged after the scans corresponds to that of the unfolding of the peptide. It DSC experiments. In the presence of peptide, lipid is partially occurs at about 65 °C for both peptides; with 3F the transition solubilized (Fig. 5). A larger fraction of cholesterol is solubi- 51408 Membrane Interactions of 3F Analogs FIG.4. Temperature dependence of the mean residue ellipticity at 222 nm of 3F-2 (left)or3F (right)in buffer and in the presence of lipid as indicated. For each run there is a heat- ing cycle (f) and a cooling cycle (). A,1 mM peptide in 20 mM phosphate buffer, 0.14 M NaCl, 1 mM EDTA, pH 7.4. B,in the presence of small unilamellar vesicles of1mM SOPC with 15% peptide. C,inthe presence of small unilamellar vesicles of 1 mM SOPC:cholesterol (1:1) with 15% pep- tide. A 1-mm cell was used with a heating scan rate of 2 K/min. lized, compared with SOPC in samples containing 0.3 mol in peptide concentration (Fig. 7). Trp emission maximum is not fraction cholesterol. It was confirmed by PAGE that lipoprotein sensitive to the presence of cholesterol, indicating that the particles were formed after either a mixture of SOPC and polarity of the Trp environment is not greatly altered. This cholesterol at a molar ratio of 1:1 or 7:3 and also containing 15 behavior is quite similar to that found with 4F (7). 3F has mol % of 3F-2 or 3F was incubated at room temperature for almost identical emission intensity in the presence of SOPC 3 h and then centrifuged. Particles of 100 Å size were seen either with or without cholesterol (Fig. 7), suggesting that this (Fig. 6), confirming that soluble lipoprotein particles were peptide does not preferentially interact with cholesterol-de- formed with both peptides. The fact that the major fraction of pleted domains. The blue shift caused by the addition of the both lipid and peptide is in the insoluble fraction at the low lipid is somewhat greater with 3F than with 3F-2, suggesting concentrations used for DSC indicates that in the case of the that the Trp of the former peptide is more deeply buried in much higher concentrations used for NMR, the major fraction the membrane. of peptide and lipid are found in the pellet and not in solubi- We have also determined the effect of cholesterol on the red lized micellar form. This is in agreement with our finding of a edge excitation shift. In accord with our previous findings (5), bilayer shaped static P NMR powder pattern for this lipid there is no red edge excitation shift with 3F-2, but there is with mixture (see below). 3F , indicating that Trp residues are more rigid with 3F-2 in Tryptophan Fluorescence—The fluorescence emission spec- presence of cholesterol containing membranes compared with 14 14 tra of 3F-2 and 3F were measured in buffer and in the 3F . It should be noted that 3F-2 has Trp at the center of the presence of lipid (Fig. 7). The emission maximum of 3F-2 in nonpolar face, whereas Leu appears at the center of the non- 14 14 buffer is 336 nm compared with 338 nm for 3F in buffer. In polar face of in 3F . This could be interpreted as a consequence the presence of lipid, either with or without cholesterol, the of Trp tending to push the peptide up toward the lipid water emission is close to 333 nm. The results indicate that the Trp interface, whereas Leu at the center of the nonpolar face in- residue inserts into the bilayer. These values are somewhat creases interaction with the lipid acyl chain and hence results blue-shifted compared with our previous study, but in a differ- in a deeper penetration into phospholipid bilayer. Cholesterol ent lipid system and at a somewhat higher peptide concentra- has no effect on this phenomenon (Fig. 8). tion. In addition, there may be some time-dependent changes Excimer Formation in the Fluorescent Probe PC P—The I /I 3 e m accounting for part of the difference. There is an 2-fold in- ratio of PC P (1% of lipid) was determined in LUVs of POPC crease in the emission intensity of 3F-2 in the presence of and POPC:cholesterol (1:1) with and without the addition of SOPC without cholesterol, which is smaller in the presence of 3F-2 or 3F at a 10:1 lipid to peptide ratio (Fig. 9). In agree- 1:1 SOPC:cholesterol. This could be a result of 3F-2 inserting ment with previous results (12), in the present work we also more deeply into bilayers not containing cholesterol, but there find that cholesterol markedly lowers the I /I ratio. This is e m may also be contributions from scattering or small differences likely a consequence of the lower rate and extent of molecular Membrane Interactions of 3F Analogs 51409 FIG.6. PAGE of supernatant fractions after centrifugation of mixtures of cholesterol, SOPC, and peptide that had been incu- bated three h at room temperature. 3 g were loaded on a 4–20% gel (PAGE) and run under native conditions for 20 h. The gel was stained with colloidal blue and destained with water. Lane 1, calibra- tion markers; lane 2, SOPC:cholesterol (1:1) with 15 mol % 3F-2; lane 3, SOPC:cholesterol (1:1) with 15 mol % 3F ; lane 4, SOPC:cholesterol (7:3) with 15 mol % 3F-2; lane 5, SOPC:cholesterol (7:3) with 15 mol % 3F . that on the diagonal (chemical shift of the slice), are clearly observed with 3F-2. This indicates that the aromatic reso- nances are in close proximity, likely as a result of stacking of the aromatic groups, both in bilayers of POPC (Fig. 10) as well as with a 1:1 mixture of POPC and cholesterol (Fig. 11). The P NMR powder pattern of 3F-2 with a 1:1 mixture of POPC and cholesterol showed only a minor isotropic component for this sample. The NOESY slices with 3F-2, for the samples with cholesterol, generally have somewhat larger cross-peaks. This is particularly evident for the cross-peaks to the acyl CH group in the spectrum with 300 ms mixing time (Fig. 11). We performed a similar analysis with the 3F peptide. The FIG.5. Solubilization of peptide and lipid in mixtures of 3F-2 31 static P NMR powder patterns for this peptide were similar to (open bars)or3F (striped bars) and multilamellar vesicle of those for 3F-2 showing a bilayer shape pattern with a signifi- SOPC with 0.3, 0.4, or 0.5 mol fraction cholesterol, each contain- cant isotropic component in the absence of cholesterol but only ing 5, 10, or 15 mol % peptide, subsequent to the series of DSC runs such as those shown in Fig. 2. Top panel, % peptide in super- a minor isotropic peak in the equimolar mixture of POPC and natant. Middle panel, % lipid in supernatant. Bottom panel, % choles- cholesterol. The slices for the sample of 3F with POPC alone terol in supernatant. (Fig. 12) generally had cross-peaks of positive sign (i.e. negative NOE), opposite to that with 3F-2. In addition, only the peak on motion in the presence of cholesterol decreasing the rate of the diagonal was observed in the aromatic region, indicating conformational change in the fluorescent probe. The effect of that the aromatic groups are less stacked in 3F than in 3F-2, the peptide is smaller and tends to increase with higher con- as might be anticipated on the basis of the larger cluster of centrations of probe, suggesting that there are both inter- and aromatic residues seen in the helical wheel projection of 3F-2 intra-molecular formation of excimers. This ratio is insensitive (Fig. 1). The cross-peaks between the lipid and 3F are par- to the presence of 3F but is affected by 3F-2, indicating that ticularly weak in the presence of cholesterol (Fig. 13), suggest- 3F-2 has a greater effect on hydrocarbon packing and/or ing that this peptide is largely excluded from cholesterol-con- dynamics. taining membranes. In addition, the aromatic region of the H NOESY MAS NMR—Slices from the two-dimensional one-dimensional spectrum is particularly well resolved com- NOESY spectrum of 3F-2 in the presence of POPC, at a 1:10 pared with other cases. peptide to lipid ratio, are presented for the spectral region of Interaction of the peptide with lipid can also be assessed by the aromatic side chain resonances using 50- or 300-ms mixing monitoring the changes in the chemical shifts of the lipid res- times (Fig. 10). The P NMR powder pattern demonstrated onances on introduction of the peptide (Table III). These that the major fraction of the lipid was in a bilayer arrange- changes in chemical shifts may arise from ring current effects ment, although there was a significant isotropic component in as well as from changes in the polarity of the environment. In the spectra of both peptides in the presence of POPC. There are the case of H MAS/NMR, only changes in the spectrum of the cross-peaks between the aromatic resonance and the protons phospholipid can be assessed, because resonances are not ob- from the lipid, particularly those of the CH protons, indicating served from protons of cholesterol in these lipid mixtures (13), insertion of the peptide into pure POPC membranes. The sign and the lower concentration of peptide makes it difficult to of the nuclear Overhauser enhancement with 50-ms mixing discern its resonances. The addition of 3F-2 or 3F to either time is negative, corresponding to rapid molecular motion, on POPC or POPC:cholesterol (1:1) results in a small change in H this time scale, between the peptide and lipid. In addition, for chemical shifts of several resonances. The change is insensitive several of the slices, peaks in the aromatic region, in addition to to the presence of cholesterol and does not indicate a preferen- 51410 Membrane Interactions of 3F Analogs FIG.7. Fluorescence emission spec- tra peptides with and without choles- terol. The left panel is for 3F , and the right panel is for 3F-2. Curve 1,15 M peptide in buffer. Curve 2,15 M peptide mixed with SOPC at a lipid to peptide ratio of 6; Curve 3,15 M peptide mixed with SOPC:cholesterol (1:1) at a lipid to peptide ratio of 6. Fluorescence intensi- ties normalized to make the emission maximum for peptide in buffer equal to 1.0. The spectra were acquired using an excitation wavelength of 280 nm at a tem- perature of 25 °C. FIG.9. Fluorescent properties of PC P at 1 mol % in LUVs of POPC or POPC-cholesterol (1:1) (lipid to peptide ratio  10). The peptide concentration was 24 M. The experiments were done at room temperature using an excitation wavelength of 344 nm. Excimer emis- sion intensity (I ) was read at 476 nm, and monomer emission intensity (I ) was read at 389 nm. contained in exchangeable plasma apolipoproteins, i.e. class A amphipathic helices (17). Although the position of the Trp residue in the two peptides is different, the nature and location of the other residues on the hydrophilic face of the helical conformation of these two peptides are identical, as is their FIG.8. The red edge excitation shift. The effect of excitation on total amino acid composition. The HPLC elution profile and the emission maximum of the intrinsic fluorescence of Trp. Upper panel, monolayer collapse pressure indicate that the two peptides 3F-2; lower panel,3F . f, peptide in PIPES buffer, pH 7.4 (30 M); , peptide in 1 mM SOPC with 15 mol % peptide; , peptide in 1 mM have similar hydrophobicities, but the 3F-2 is somewhat less SOPC:cholesterol (1:1) with 15 mol % peptide. The fluorescence was hydrophobic (5). Both peptides can rapidly solubilize 1-palmi- measured at 25 °C. toyl-2-oleoyl phosphatidylcholine at an equimolar ratio of pep- tide and lipid (5). The CD spectra of the two peptides are tial location of the peptide in the bilayer. Because cholesterol similar, and we show in this work that the secondary structure resonances are not observed in H MAS/NMR, we also meas- 13 is independent of the presence of cholesterol (Fig. 3). We also ured C MAS/NMR. The changes in chemical shift are gener- show that both peptides undergo a loss of secondary structure ally small, and there is no large difference between 3F-2 and 14 on heating (Fig. 4). However, the thermal denaturation of 3F-2 3F . In mixtures containing cholesterol the changes in chem- is more prominent in DSC scans of 3F-2 than of 3F (Fig. 2). ical shifts are comparable for POPC and for cholesterol (not Because a similar amount of helicity is lost upon heating of the shown). two peptides, we suggest that the transition of 3F-2 is more DISCUSSION cooperative because this peptide has a higher degree of oli- Using in vitro assays expected to correlate with protection gomerization on the membrane surface compared with 3F . against atherosclerosis, 3F-2 and 3F exhibit quite different We have shown that another biologically potent class A hel- potency (5); however, the differences in their biophysical prop- ical peptide, 4F, promoted the formation of cholesterol-rich erties have been found to be relatively small (this work and Ref. domains by preferentially interacting with the phospholipid 5). Both peptides are class A amphipathic helices and therefore component of cholesterol/SOPC mixtures (7). This finding pro- can fold into a helical structure resembling amphipathic helices vided an interesting contrast with the peptide LWYIK that Membrane Interactions of 3F Analogs 51411 FIG. 10. One-dimensional slices from the MAS H NOESY spectrum at the chemical shifts of the aromatic protons of a sample of POPC containing 10 mol % 3F-2. The mixing times were 50 ms (left panel) and 300 ms (right panel). The top spectra are conventional one-dimensional proton spectra of the samples. The resonance assignments are indicated on the top spectrum. FIG. 11. One-dimensional slices from the MAS H NOESY spectrum at the chemical shifts of the aromatic protons of a sample of POPC:cholesterol (1:1) containing 10 mol % 3F-2. The mixing times were 50 ms (left panel) and 300 ms (right panel). The top spectra are conventional one-dimensional proton spectra of the samples. The resonance assignments are indicated on the top spectrum. preferentially interacted with cholesterol (16). Rearrangement than are required for their formation in the absence of peptide of cholesterol in membranes can result from preferential inter- (Fig. 2 and Table I). The peptide 3F also promotes the forma- action of proteins with cholesterol-rich domains as well as with tion of cholesterol crystals. However, 3F does not lower the cholesterol-depleted domains (18). These observations are enthalpy of the phase transition of SOPC in mixtures with likely to be relevant to the mechanism of formation of “rafts” in cholesterol (Table II), indicating that it is not interacting pref- biological membranes. The DSC shows that 3F-2 interacts pref- erentially with SOPC. 3F also does not greatly increase the erentially with the phospholipid in mixtures of cholesterol and enthalpy of SOPC by removing cholesterol, as we had shown for SOPC. This is indicated by the fact that this peptide is much LWYIK (16). We suggest that a contributing factor to the pro- more potent in lowering the phase transition of SOPC than is motion of cholesterol segregation in the membrane is through 3F (Fig. 2 and Table II). 3F-2 also promotes the formation of an increase in the lateral pressure of the membrane “squeezing cholesterol crystals at mol fractions of cholesterol much lower out” cholesterol. The greater preference of 3F-2 for cholesterol- 51412 Membrane Interactions of 3F Analogs FIG. 12. One-dimensional slices from the MAS H NOESY spectrum at the chemical shifts of the aromatic protons of a sample of POPC containing 10 mol % 3F . The mixing times were 50 ms (left panel) and 300 ms (right panel). The top spectra are conventional one-dimensional proton spectra of the samples. The resonance assignments are indicated on the top spectrum. FIG. 13. One-dimensional slices from the MAS H NOESY spectrum at the chemical shifts of the aromatic protons of a sample of POPC:cholesterol (1:1) containing 10 mol % 3F . The mixing times were 50 ms (left panel) and 300 ms (right panel). The top spectra are conventional one-dimensional proton spectra of the samples. In the case of this figure the one-dimensional spectrum was more highly expanded to show the clearly resolved weak aromatic peaks. The resonance assignments are indicated on the top spectrum. depleted domains is also common to another biologically potent There is also a difference between the two peptides in how peptide of this series, 4F (7). This correlation is not seen with they interact with the membrane. The emission intensity from the less biologically active peptide 3F . the Trp of 3F-2 is increased more by lipid, especially the lipid Membrane Interactions of 3F Analogs 51413 TABLE III Changes in H chemical shifts of lipid resonances induced by 3F peptides Chemical shift difference With 3F-2 With 3F Resonance POPC/cholesterol POPC/cholesterol POPC POPC (1:1) (1:1) ppm Glycerol C2 0.04 0.04 0.05 0.02 Glycerol C3 0.03 0.03 0.02 0.02 Choline  0.03 0.03 0.03 0.03 Glycerol C1 0.03 0.04 0.03 0.03 Choline  0.03 0.03 0.02 0.05 Quaternary CH 0.03 0.03 0.02 0.02 CH CO 0.01 0.03 0 0.03 CH CCO 0.03 0.04 0.02 0.07 CH 0.03 0.02 0.02 0.01 Terminal CH 0.03 0.02 0.03 0.02 Chemical shift differences are in parts per million between that of lipid alone and in the presence of 10 mol% peptide. A positive charge corresponds to a shift in the resonance to a lower frequency caused by FIG. 14. Biological activity of a Class A amphipathic helix de- the peptide. pends on hydrophobic face-lipid acyl chain interaction. Top panel, a minimal effect on lipid acyl chain packing occurs in the wedge- 14 14 shaped molecule 3F . Bottom panel, the cylindrical shaped peptide, mixture containing cholesterol (Fig. 7), than is the case of 3F . 3F-2, causes greater acyl chain perturbations, facilitating the entry of The behavior of the Trp fluorescence of 3F-2 is qualitatively molecules such as water and lipid hydroperoxides into the hydrophobic similar to that of 4F (7), even though the Trp is in different milieu of the complex. positions for the two peptides. The similar behavior of the Trp fluorescence of two peptides, despite the different position of Trp in the helical wheel, suggests that 3F penetrates more The NMR parameters that we have monitored are not very deeply into the bilayer. The greater number of cross-peaks sensitive to the hydrocarbon packing or molecular motion in between protons of the aromatic residues of 3F-2 (Fig. 10) the interior of the membrane. We have therefore also studied indicates that its side chains are stacked in the presence of the properties of the fluorescent probe PC P that has been lipid. In the presence of cholesterol particularly, 3F-2 exhibits suggested to monitor changes in membrane motional proper- several strong, negative cross-peaks with several lipid protons, ties (12, 19). We do not wish to use the probe as the basis for a including those from the terminal CH group of the acyl chain. model of the interaction of the peptide with lipid but rather as The peaks of negative sign of the NOESY suggests increased a demonstration that the more active peptide, 3F-2, has a much molecular motion that could allow transient access to the pro- larger effect on the packing properties of the hydrocarbon re- tons in the center of the bilayer. This conclusion appears oppo- gion of the membrane, compared with the less active 3F . The site to that derived from red edge excitation shift, indicating a effects are consistent with the proposed model for the interac- restricted motion of the Trp of 3F-2. We suggest that this tion of these peptides with bilayers based on molecular shape difference is a consequence of the widely different time scale for (Fig. 14). In this model the 3F-2 peptide would introduce the two measurements. Fluorescence decay occurs in nanosec- greater destabilization of the bilayer as a consequence of its onds, whereas proton relaxation occurs in milliseconds to sec- cylindrical shape, short length, and aromatic side chains push- onds. Hence in the longer time scale of NMR, the relative ing the peptide axis up closer to the polar-nonpolar interface. position between the Trp and the lipid can change, but locally Accumulation of the probe in the bilayer defect would dilute the around the Trp the environment is rigid, and there is slow PC P and decrease intermolecular excimer formation and pos- reorientation of the surrounding dipoles. According to the sibly also disfavor the conformation required for pyrene dimer- model we proposed (Fig. 14), 3F-2 would, because of its cylin- ization in the monomer. This is also consistent with the greater drical shape, short length, depth of penetration, and aromatic anti-atherogenic properties of 3F-2, because decreased mem- side chains pushing the peptide axis up closer to the polar- brane order has been suggested to be associated with increased nonpolar interface, introduce a destabilization in the bilayer risk for cardiovascular disease (20). resulting in a decrease in the lipid order parameters. The In summary, given the marked differences in biological ac- 14 14 decreased penetration of 3F-2 compared with 3F is also indi- tivity between 3F-2 and 3F , the nature of the interaction of cated by the small shift in Trp emission wavelength caused by these peptides with SOPC with or without cholesterol is re- lipid (Fig. 7). Again there is a difference between peptide pen- markably similar. Nevertheless there are important differ- etration into the membrane as assessed by NOE effects and by ences between the two peptides that support our model of the fluorescence. It is possible that the greater bilayer disruption difference in peptide “shape” (5). Although 3F-2 is less hydro- caused by 3F-2 allows some penetration of water into the mem- phobic than 3F by the criteria of HPLC volume and mono- brane, resulting in a smaller effect on Trp emission, even layer exclusion pressure (5), 3F-2 has stronger NOESY cross- though the NOSEY spectra indicate a greater penetration of peaks with protons more in the center of the bilayer (Fig. 10). this peptide. In comparison, 3F appears to be largely ex- We suggest that this is a consequence of a greater disordering cluded from bilayers containing cholesterol. It shows only very of the bilayer caused by this peptide. 3F-2 also has two features weak cross-peaks between the aromatic resonances and the that are more in common with those of 4F (7). These are a lipid protons (Fig. 11). This is also in accord with the smaller preferential broadening of the chain melting transition in mix- change in the intensity of emission from the Trp residue in the tures of SOPC and cholesterol (Fig. 2) and a more cooperative presence of cholesterol. However, when 3F incorporates into unfolding transition of the peptide. The latter suggests oli- the bilayer, it may be able to pack with the acyl chains with less gomerization that may also contribute to a greater disruption disruption of the hydrocarbon portion of the bilayer, as sug- of the bilayer order by insertion of a larger peptide aggregate. gested in Fig. 14. The disordering of the lipid could allow for the transfer of 51414 Membrane Interactions of 3F Analogs (2004) J. Biol. Chem. 279, 26509–26517 oxidized lipids from the LDL surface to peptide-containing 6. Ou, Z., Ou, J., Ackerman, A. W., Oldham, K. T., and Pritchard, K. A., Jr. (2003) particles, thus rendering LDL less effective in inducing mono- Circulation 107, 1520–1524 7. Epand, R. M., Epand, R. F., Sayer, B. G., Melacini, G., Palgulachari, M. N., cyte chemotaxis, an important step in the initiation Segrest, J. P., and Anantharamaiah, G. M. (2004) Biochemistry 43, of atherogenesis. 5073–5083 8. Datta, G., Chaddha, M., Hama, S., Navab, M., Fogelman, A. M., Garber, D. W., Acknowledgment—We are grateful to Martin Jones for help with Mishra, V. K., Epand, R. M., Epand, R. F., Lund-Katz, S., Phillips, M. C., the figures. Segrest, J. P., and Anantharamaiah, G. M. (2001) J. Lipid Res. 42, 1096–1104 REFERENCES 9. Ames, B. N. (1966) Methods Enzymol. 8, 115–118 10. Zlatkis, A., Zak, B., and Boyle, A. J. (1953) J. Lab. Clin. Med. 41, 486–492 1. Navab, M., Anantharamaiah, G. M., Reddy, S. T., Van Lenten, B. J., Hough, 11. Laemmli, U. K. (1970) Nature 227, 680–685 G., Wagner, A., Nakamura, K., Garber, D. W., Datta, G., Segrest, J. P., 12. Melnick, R. L., Haspel, H. C., Goldenberg, M., Greenbaum, L. M., and Wein- Hama, S., and Fogelman, A. M. (2003) Curr. Opin. Investig. Drugs 4, stein, S. (1981) Biophys. J. 34, 499–515 1100–1104 13. Forbes, J., Bowers, J., Shan, X., Moran, L., Oldfield, E., and Moscarello, M. A. 2. Garber, D. W., Datta, G., Chaddha, M., Palgunachari, M. N., Hama, S. Y., (1988) J. Chem. Soc. Faraday Transactions 84, 3821–3849 Navab, M., Fogelman, A. M., Segrest, J. P., and Anantharamaiah, G. M. 14. Guo, W., and Hamilton, J. A. (1996) Biophys. J. 71, 2857–2868 (2001) J. Lipid Res. 42, 545–552 15. Arnold, M. R., Kremer, W., Ludemann, H. D., and Kalbitzer, H. R. (2002) 3. Navab, M., Anantharamaiah, G. M., Hama, S., Garber, D. W., Chaddha, M., Biophys. Chem. 96, 129–140 Hough, G., Lallone, R., and Fogelman, A. M. (2002) Circulation 105, 290–292 16. Epand, R. M., Sayer, B. G., and Epand, R. F. (2003) Biochemistry 42, 14677–14689 4. Navab, M., Anantharamaiah, G. M., Reddy, S. T., Hama, S., Hough, G., Grijalva, V. R., Wagner, A. C., Frank, J. S., Datta, G., Garber, D., and 17. Anantharamaiah, G. M., Jones, M. K., and Segrest, J. P. (1993) in The Am- phipathic Helix (Epand, R. M., ed) pp 109–142, CRC Press, Boca Raton, FL Fogelman, A. M. (2004) Circulation 109, 3215–3220 5. Datta, G., Epand, R. F., Epand, R. M., Chaddha, M., Kirksey, M. A., Garber, 18. Epand, R. M. (2004) Biochim. Biophys. Acta 1666, 227–238 D. W., Lund-Katz, S., Phillips, M. C., Hama, S., Navab, M., Fogelman, 19. Hartel, S., Diehl, H. A., and Ojeda, F. (1998) Anal. Biochem. 258, 277–284 A. M., Palgunachari, M. N., Segrest, J. P., and Anantharamaiah, G. M. 20. Tabas, I. (2002) J. Clin. Invest. 110, 905–911 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Two Homologous Apolipoprotein AI Mimetic Peptides

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 49, Issue of December 3, pp. 51404–51414, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. RELATIONSHIP BETWEEN MEMBRANE INTERACTIONS AND BIOLOGICAL ACTIVITY* Received for publication, July 28, 2004, and in revised form, September 8, 2004 Published, JBC Papers in Press, September 8, 2004, DOI 10.1074/jbc.M408581200 Richard M. Epand‡§, Raquel F. Epand‡, Brian G. Sayer‡, Geeta Datta¶, Manjula Chaddha¶, and G. M. Anantharamaiah¶ From the ‡Departments of Biochemistry and Biomedical Sciences and Chemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada and the ¶Departments of Medicine, Biochemistry, and Molecular Genetics and the Atherosclerosis Research Unit, University of Alabama at Birmingham, Birmingham, Alabama 35294 Two related 18-amino acid, class A, amphipathic heli- density lipoprotein-like particles containing peptide, choles- cal peptides termed 3F-2 and 3F were chosen for this terol, apo A-I and paraoxonase, an enzyme capable of convert- study. Although they have identical amino acid compo- ing pro-inflammatory high density lipoprotein into anti-inflam- sitions and many similar biophysical properties, 3F-2 is matory high density lipoprotein (4). The peptide 4F has also more potent than 3F as an apolipoprotein AI mimetic been tested in vitro and shown to effectively inhibit lytic pep- peptide. The two peptides exhibit similar gross confor- tide-induced hemolysis, inhibit oxidized phospholipid-induced mational properties, forming structures of high helical monocyte chemotaxis, scavenge lipid hydroperoxides from LDL content on a membrane surface. However, the thermal (5), and maintain endothelial nitric-oxide synthetase activity in denaturation transition of 3F-2 is more cooperative, sug- the presence of atherogenic concentrations of LDL (6). To study gesting a higher degree of oligomerization on the mem- the relationship of peptide structure to anti-atherosclerogenic brane. Both 3F-2 and 3F promote the segregation of potency, we studied the properties of four related 18-amino cholesterol in membranes containing phosphatidylcho- acid, class A amphipathic helical peptides (5). All of these line and cholesterol, but 3F-2 exhibits a greater selectiv- peptides had identical amino acid compositions and very sim- ity for partitioning into cholesterol-depleted regions of ilar physical properties, yet two of these peptides, 3F-1 and the membrane. Magic angle spinning/NMR studies indi- 3F-2, were more potent in inhibiting lytic peptide-induced he- cate that the aromatic residues of 3F-2 are stacked in the molysis, inhibiting oxidized phospholipid-induced monocyte presence of lipid. The aromatic side chains of this pep- chemotaxis, and scavenging lipid hydroperoxides from LDL com- tide also penetrate more deeply into membranes of 3 14 pared with the analogs 3F and 3F (5). In the present work we phosphatidylcholine with cholesterol compared with compare the interaction with phospholipid bilayers with and 3F . Using the fluorescent probe, 1,3-dipyrenylpropane, we monitored the properties of the lipid hydrocarbon without cholesterol, of the most potent peptide of this series, 3F-2 environment. 3F-2 had a greater effect in altering the (Ac-DKWKAVYDKFAEAFKEFL-NH ), and the least potent properties of the hydrocarbon region of the membrane. among these peptides, 3F (Ac-DWLKAFYDKVAEKFKEAF- The results are consistent with our proposed model of NH ) (Fig. 1). We have previously shown that the potent analog the effect of peptide shape on the nature of the differ- 4F that has four, rather than three Phe, is capable of forming ence in peptide insertion into the bilayer. cholesterol-rich domains by preferentially interacting with re- gions of the membrane that are depleted of cholesterol (7). 3F-2 exhibits similar biological potency to 4F but has somewhat less There is growing evidence that certain apo A-I mimetic, activity in inhibiting oxidized phospholipid-induced monocyte class A amphipathic helical peptides can be used to inhibit chemotaxis, about the same activity in scavenging lipid hy- atherosclerosis (1). The oral administration of peptide 4F syn- droperoxides from LDL and greater activity in inhibiting lytic thesized from all-D amino acids (D-4F) protects mice from peptide-induced hemolysis (5). In contrast, although 3F is a diet-induced atherosclerosis without altering plasma choles- class A amphipathic helical peptide with some anti-atherosclero- terol levels (2, 3). Preliminary studies also suggest that oral genic activity, it has a much weaker potency than either 3F-2 or administration of D-4F to LDL receptor null and apo E null 4F in the activities mentioned. This difference in potency is mice causes the rapid formation and clearance of small high reflected in differences in the red edge effect in Trp emission from the peptide and in the quenching of 2-(3 (diphenylhexatrienyl)- propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (5). In * This work was supported by Grant MT-7654 from the Canadian this study we further evaluate the interaction of 3F-2 and 3F Institutes of Health Research and Grants HL 34343 and RO1 HL 65663 with model membranes using NMR, DSC, CD, and fluorescence. from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance MATERIALS AND METHODS with 18 U.S.C. Section 1734 solely to indicate this fact. Lipids—The lipids used in this study were obtained from Avanti § To whom correspondence should be addressed. E-mail: epand@ Polar Lipids (Alabaster, AL). The purity of the phospholipids was ver- mcmaster.ca. 1 ified by measuring the cooperativity and temperature of the phase The abbreviations used are: apo, plasma apolipoprotein; LDL, low transition using DSC. density lipoprotein; PC, phosphatidylcholine; PO, 1-palmitoyl-2-oleoyl; Peptide Synthesis—The peptides were synthesized by the solid phase SO, 1-stearoyl-2-oleoyl; PC P, 1,3-dipyrenylpropane; MAS, magic angle method with a Protein Technologies PS-3 automatic peptide synthe- spinning; NOESY, nuclear Overhauser enhancement spectroscopy; sizer using the procedures described previously (2, 8). The peptides LUV, large unilamellar vesicle; DSC, differential scanning calorimetry; were purified using a preparative HPLC system (Beckman Gold), and HPLC, high pressure liquid chromatography; PIPES, 1,4-piperazinedi- ethanesulfonic acid; I , intensity of excimer emission; I , intensity of the purity of the peptides was ascertained by mass spectral analysis e m monomer emission. and analytical HPLC. 51404 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Membrane Interactions of 3F Analogs 51405 FIG.1. Helical wheel representa- tion of 3F-2 and 3F and molecular models of these peptides with sur- rounding phosphatidylcholine. The wheel is projected along the axis of the helix from the N to the C terminus with the hydrophobic side facing downward. The primary structure is given above each wheel diagram. The amino acid composi- tion of both peptides is the same. The sequence is different. The plus and minus signs denote the charges on the amino acids at neutral pH. The red color denotes an acidic residue, the blue color denotes a basic residue, and the bold black denotes aromatic residues. Concentrations of Peptide and Lipid—The concentrations of peptide Temperature heating or cooling scans were performed at a rate of solutions in buffer were determined spectrophotometrically using the 2 °C/min, with a 30-s equilibration pause at each point for thermal 1 1 absorbance at 280 nm and an extinction coefficient of 6970 cm M , equilibration. Ellipticity at 222 nm was measured as a function of calculated from the Tyr and Trp content. Phospholipid concentration temperature between 25 and 95 °C and then cooling back to 25 °C to was determined by phosphate analysis (9). assess the reversibility of denaturation. Preparation of Samples for DSC and NMR Experiments—Phospho- Large Unilamellar Vesicles (LUVs)—Lipid films were made by dis- lipid and cholesterol were dissolved in chloroform/methanol (2/1, v/v). solving appropriate amounts of lipid in a mixture of chloroform/meth- For samples containing peptide, an aliquot of a solution of the peptide anol (2/1, v/v) and dried in a test tube under nitrogen to deposit the lipid in methanol was added to the lipid solution in chloroform/methanol. as a thin film on the wall of the tube. Final traces of solvent were The solvent was then evaporated under a stream of nitrogen with removed in a vacuum chamber attached to a liquid nitrogen trap for 2–3 constant rotation of a test tube so as to deposit a uniform film of lipid h. Dried films were kept under argon gas at 30 °C if not used imme- over the bottom third of the tube. The last traces of solvent were diately. The films were hydrated with buffer, vortexed extensively at removed by placing the tube under high vacuum for at least 2 h. The room temperature, and then subjected to five cycles of freezing and lipid film was then hydrated with 20 mM PIPES, 1 mM EDTA, 150 mM thawing. The homogeneous lipid suspensions were then further pro- NaCl with 0.002% NaN , pH 7.40, and suspended by intermittent cessed by 10 passes through two stacked 0.1-m polycarbonate filters vortexing and heating to 50 °C for 2 min under argon. The samples used (Nucleopore Filtration Products, Pleasanton, CA) in a barrel extruder for NMR analysis were hydrated with the same buffer made in H O (Lipex Biomembranes, Vancouver, Canada), at room temperature. adjusted to a pH meter reading of 7.0 (pD  7.4). The samples used for LUVs were kept on ice and used within a few hours of preparation. NMR were incubated 24 h at 4 °C to allow conversion of anhydrous Tryptophan Fluorescence—Fluorescence emission spectra of the pep- cholesterol crystals to the monohydrate form. For the NMR measure- tides in the presence and absence of LUVs were measured at 25 °C ments, the samples were spun in an Eppendorf centrifuge at room using an SLM Aminco Series II luminescence spectrometer. The exci- temperature. The resulting hydrated pellet was transferred to a 12-l tation wavelength was varied between 280 and 310 nm. The emission capacity Kel-F spherical insert of an 18  4-mm ZrO rotor, attempting 2 scans were recorded and then corrected for inner filter and instrumen- to pack the maximal amount of lipid into the rotor while maintaining tal effects. it wet. Excimer Formation in the Fluorescent Probe PC P—The physical Differential Scanning Calorimetry—Measurements were made im- properties of the hydrophobic region of the membrane and the effects of mediately after sample preparation using a Nano Differential Scanning the 3F peptides were monitored by measuring the fluorescent emission Calorimeter (Calorimetry Sciences Corporation, American Fork, UT). of PC P (Molecular Probes, Eugene, OR). It has been shown that the The scan rate was 2 °C/min, and there was a delay of 5 min between ratio of the intensities of excimer and monomer emission is sensitive to sequential scans in a series to allow for thermal equilibration. DSC the “fluidity” of the membrane interior (12). LUVs were prepared con- curves were analyzed by using the fitting program, DA-2, provided by taining lipids and PC P at a 100:1 molar ratio in Hepes buffer (10 mM Microcal Inc. (Northampton, MA) and plotted with Origin, version 5.0. Hepes, 0.14 M NaCl, 1 mM EDTA, pH 7.4). Florescence measurements Centrifugation Assay for Membrane Binding of 3F Peptides—The were made in 1  1-cm-square quartz cuvettes containing 2 ml of Hepes fraction of peptide bound to the lipid after three heating and cooling buffer at room temperature. The excitation wavelength was 344 nm cycles between 0 and 100 °C was determined by separating the free using slits with 4-nm band pass in both emission and excitation. Emis- peptide by centrifugation. The vesicles with bound protein were pel- sion scans were made between 360 and 550 nm before and after the leted at 200,000  g for 90 min at 25 °C. A clear supernatant was addition of peptide. The results are summarized as the ratio of the separated from the solid pellet and assayed for protein by absorption at intensity of excimer emission at 476 nm to that of monomer emission at 278 nm. The absorption of a blank was subtracted, and a base line, set 389 nm (I /I ). The experiments were repeated twice with two inde- e m at 350 nm for each sample, was used to correct for residual scattering. pendent preparations. The error in I /I for duplicate measurements e m In addition, the amount of lipid in both pellet and supernatant fractions with the same preparations was between 1 and 2%, but between inde- was determined by phosphate analysis. Cholesterol concentration was pendently prepared samples the absolute value of the ratios varied determined by a modified version of the procedure of Zlatkis et al. (10). between 5 and 10%; however, they always exhibited similar relative Briefly, after extraction with chloroform, the organic phase was dried values with and without either of the two peptides. under nitrogen and then placed under vacuum to completely remove the 1 H NOESY MAS NMR—High resolution MAS spectra were acquired solvent. To the dried samples or to the dry cholesterol standards, glacial using a spinning rate of 4 kHz in a Bruker DRX 500 NMR spectrometer. acetic acid was added, followed by a reagent composed of FeCl and 85% Probe temperature was 24  1 °C. The two-dimensional NOESY spec- H PO in sulfuric acid. The samples were placed in a boiling water bath 3 4 tra were obtained using mixing times of 50 and 300 ms. The resonances for 3 min to develop a purple color. After cooling the absorbance was were assigned based on their close similarity to literature values for read at 550 nm. phosphatidylcholine (13), cholesterol (14), and amino acid residues (15). To test for the presence of lipoprotein particles in the supernatant, it was analyzed by nondenaturing PAGE using the method of Laemmli RESULTS (11). An aliquot of the supernatant containing 3 g of peptide was loaded on a 4–20% gel and run under native conditions for 20 h. The gel DSC—Lipid mixtures of SOPC containing 0, 0.3, 0.4, and 0.5 was then stained with colloidal blue and destained with water. mol fractions cholesterol were analyzed by DSC in the presence Circular Dichroism—CD spectra were recorded on an AVIV model of 0, 5, 10, and 15 mol % 3F-2 or 3F . As examples, we present 215 spectropolarimeter using a quartz cell with a 0.1-cm path length. the results from mixtures containing 0 or 15 mol % of each of The cuvette was placed in a jacketed cell holder maintained at the the peptides (Fig. 2). The DSC curves are presented as the desired temperature with circulating thermostatic fluid. The lipid was excess heat capacity/mol cholesterol as in our earlier paper on solubilized by incubation with the peptide followed by sonication to ensure minimal effects of light scattering. 4F (7). Both peptides clearly promote the separation of choles- 51406 Membrane Interactions of 3F Analogs FIG.2. Differential scanning calorimetry of SOPC:cholesterol mixtures with 0, 5, 10, or 15 mol % 3F-2 or 3F . Scan rate 2 K/min. The rows correspond to lipid compositions of 7:3 (top row), 6:4 (middle row), and 1:1 (bottom row) SOPC:cholesterol, respectively. The columns correspond to lipid alone (first column) or lipid with the addition of 15% of either 3F-2 (second column)or3F (third column). Lipid concentration is 2.5 mg/ml in 20 mM PIPES, 1 mM EDTA, 150 mM NaCl with 0.002% NaN , pH 7.40. Sequential heating and cooling scans between 0 and 100 °C. The numbers are the order in which the scans were carried out, with scans 1 and 3 being heating scans, each of which was followed by one of the cooling scans 2 or 4. Scans were displaced along the y axis for clarity of presentation. TABLE I Enthalpy of the polymorphic transition of anhydrous cholesterol crystals The values are in cal/mol cholesterol determined from areas under the transitions at 36 °C on the first DSC heating scan. 3F-2 3F Cholesterol in SOPC 510 15510 15 30% 00000 85 40% 0 170 55 55 95 160 50% 250 225 400 200 200 225 terol into crystalline domains at higher mol fractions of choles- been found previously, albeit to a lesser extent, with the pep- terol and of peptide (Table I). The cholesterol crystals formed tide 4F (7). Scanning only up to 50 °C can eliminate much of are in a metastable state and disappear after sequential heat- this loss (16), but the higher temperatures are required to ing and cooling scans between 0 and 100 °C (Fig. 2). This has observe the unfolding transition of the peptide. 3F causes Membrane Interactions of 3F Analogs 51407 TABLE II Enthalpy of the chain melting transition of SOPC The values are in cal/mol SOPC determined from the average area under the transitions at 6 °C on the DSC cooling scans. 3F-2 3F Cholesterol in SOPC 0 5 10 15 5 10 15 0% 4000 ND ND 2200 ND ND 2400 30% 500 800 345 278 515 515 530 40% 350 0 0 0 470 570 265 50% 210 0 0 0 300 220 200 ND, not determined. FIG.3. Circular dichroism spectra of 3F-2 (left) and 3F (right)in20mM phosphate buffer, 0.14 M NaCl, 1 mM EDTA, pH 7.4 (A), in the presence of small unilamellar vesicles of 1 mM SOPC with 15% peptide (B), and small unilamellar vesicles of 1 mM SOPC:cholesterol (1:1) with 15% pep- tide (C). The lipid was solubilized by the peptide, but the mixture was also soni- cated to reduce the possibility of scatter- ing artifacts. The spectra were taken in a 1-mm cell at 25 °C. separation of cholesterol crystals at lower mol fractions of cho- is readily observed only with SOPC in the absence of choles- lesterol and peptide than is required with 3F-2. A more marked terol (not shown), but the transition is quite prominent in scans difference is observed in the peak at the lowest transition with 3F-2 (Fig. 2). The transition temperature is slightly lower temperature that is ascribed to the gel to liquid crystalline on cooling than on heating. transition of SOPC. The enthalpy of this transition can be CD—The CD spectra in buffer of both 3F-2 and 3F exhibit reasonably estimated from cooling scans. For pure SOPC (data some dependence on peptide concentration (5). The addition of not shown), the transition occurs at 5.5 °C on cooling at 2 K/ SOPC or SOPC:cholesterol (1:1) to 3F-2 at 100 M peptide min with a transition enthalpy of 4 kcal/mol (16). With pure results in little change in the secondary structure of 3F-2 (Fig. SOPC without cholesterol, the addition of 15 mol % 3F-2 lowers 3) but slightly increases the magnitude of the CD for 3F (Fig. the enthalpy to 2.2 kcal/mol and to 2.4 kcal/mol with 3F (data 3). The temperature dependence of the spectrum shows a sub- not shown). With mixtures of cholesterol and SOPC the addi- stantial loss of secondary structure on heating to 95 °C with tion of increasing concentrations of 3F-2 eliminates this tran- both 3F-2 and with 3F (Fig. 4). The thermal transition is sition, whereas with 3F the enthalpy of this transition is broad with some hysteresis on heating and cooling. slightly increased (Table II). Solubilization of Lipid and Peptide—Mixtures of SOPC, cho- The highest temperature transition observed in the DSC lesterol, and the peptide 3F-2 or 3F were centrifuged after the scans corresponds to that of the unfolding of the peptide. It DSC experiments. In the presence of peptide, lipid is partially occurs at about 65 °C for both peptides; with 3F the transition solubilized (Fig. 5). A larger fraction of cholesterol is solubi- 51408 Membrane Interactions of 3F Analogs FIG.4. Temperature dependence of the mean residue ellipticity at 222 nm of 3F-2 (left)or3F (right)in buffer and in the presence of lipid as indicated. For each run there is a heat- ing cycle (f) and a cooling cycle (). A,1 mM peptide in 20 mM phosphate buffer, 0.14 M NaCl, 1 mM EDTA, pH 7.4. B,in the presence of small unilamellar vesicles of1mM SOPC with 15% peptide. C,inthe presence of small unilamellar vesicles of 1 mM SOPC:cholesterol (1:1) with 15% pep- tide. A 1-mm cell was used with a heating scan rate of 2 K/min. lized, compared with SOPC in samples containing 0.3 mol in peptide concentration (Fig. 7). Trp emission maximum is not fraction cholesterol. It was confirmed by PAGE that lipoprotein sensitive to the presence of cholesterol, indicating that the particles were formed after either a mixture of SOPC and polarity of the Trp environment is not greatly altered. This cholesterol at a molar ratio of 1:1 or 7:3 and also containing 15 behavior is quite similar to that found with 4F (7). 3F has mol % of 3F-2 or 3F was incubated at room temperature for almost identical emission intensity in the presence of SOPC 3 h and then centrifuged. Particles of 100 Å size were seen either with or without cholesterol (Fig. 7), suggesting that this (Fig. 6), confirming that soluble lipoprotein particles were peptide does not preferentially interact with cholesterol-de- formed with both peptides. The fact that the major fraction of pleted domains. The blue shift caused by the addition of the both lipid and peptide is in the insoluble fraction at the low lipid is somewhat greater with 3F than with 3F-2, suggesting concentrations used for DSC indicates that in the case of the that the Trp of the former peptide is more deeply buried in much higher concentrations used for NMR, the major fraction the membrane. of peptide and lipid are found in the pellet and not in solubi- We have also determined the effect of cholesterol on the red lized micellar form. This is in agreement with our finding of a edge excitation shift. In accord with our previous findings (5), bilayer shaped static P NMR powder pattern for this lipid there is no red edge excitation shift with 3F-2, but there is with mixture (see below). 3F , indicating that Trp residues are more rigid with 3F-2 in Tryptophan Fluorescence—The fluorescence emission spec- presence of cholesterol containing membranes compared with 14 14 tra of 3F-2 and 3F were measured in buffer and in the 3F . It should be noted that 3F-2 has Trp at the center of the presence of lipid (Fig. 7). The emission maximum of 3F-2 in nonpolar face, whereas Leu appears at the center of the non- 14 14 buffer is 336 nm compared with 338 nm for 3F in buffer. In polar face of in 3F . This could be interpreted as a consequence the presence of lipid, either with or without cholesterol, the of Trp tending to push the peptide up toward the lipid water emission is close to 333 nm. The results indicate that the Trp interface, whereas Leu at the center of the nonpolar face in- residue inserts into the bilayer. These values are somewhat creases interaction with the lipid acyl chain and hence results blue-shifted compared with our previous study, but in a differ- in a deeper penetration into phospholipid bilayer. Cholesterol ent lipid system and at a somewhat higher peptide concentra- has no effect on this phenomenon (Fig. 8). tion. In addition, there may be some time-dependent changes Excimer Formation in the Fluorescent Probe PC P—The I /I 3 e m accounting for part of the difference. There is an 2-fold in- ratio of PC P (1% of lipid) was determined in LUVs of POPC crease in the emission intensity of 3F-2 in the presence of and POPC:cholesterol (1:1) with and without the addition of SOPC without cholesterol, which is smaller in the presence of 3F-2 or 3F at a 10:1 lipid to peptide ratio (Fig. 9). In agree- 1:1 SOPC:cholesterol. This could be a result of 3F-2 inserting ment with previous results (12), in the present work we also more deeply into bilayers not containing cholesterol, but there find that cholesterol markedly lowers the I /I ratio. This is e m may also be contributions from scattering or small differences likely a consequence of the lower rate and extent of molecular Membrane Interactions of 3F Analogs 51409 FIG.6. PAGE of supernatant fractions after centrifugation of mixtures of cholesterol, SOPC, and peptide that had been incu- bated three h at room temperature. 3 g were loaded on a 4–20% gel (PAGE) and run under native conditions for 20 h. The gel was stained with colloidal blue and destained with water. Lane 1, calibra- tion markers; lane 2, SOPC:cholesterol (1:1) with 15 mol % 3F-2; lane 3, SOPC:cholesterol (1:1) with 15 mol % 3F ; lane 4, SOPC:cholesterol (7:3) with 15 mol % 3F-2; lane 5, SOPC:cholesterol (7:3) with 15 mol % 3F . that on the diagonal (chemical shift of the slice), are clearly observed with 3F-2. This indicates that the aromatic reso- nances are in close proximity, likely as a result of stacking of the aromatic groups, both in bilayers of POPC (Fig. 10) as well as with a 1:1 mixture of POPC and cholesterol (Fig. 11). The P NMR powder pattern of 3F-2 with a 1:1 mixture of POPC and cholesterol showed only a minor isotropic component for this sample. The NOESY slices with 3F-2, for the samples with cholesterol, generally have somewhat larger cross-peaks. This is particularly evident for the cross-peaks to the acyl CH group in the spectrum with 300 ms mixing time (Fig. 11). We performed a similar analysis with the 3F peptide. The FIG.5. Solubilization of peptide and lipid in mixtures of 3F-2 31 static P NMR powder patterns for this peptide were similar to (open bars)or3F (striped bars) and multilamellar vesicle of those for 3F-2 showing a bilayer shape pattern with a signifi- SOPC with 0.3, 0.4, or 0.5 mol fraction cholesterol, each contain- cant isotropic component in the absence of cholesterol but only ing 5, 10, or 15 mol % peptide, subsequent to the series of DSC runs such as those shown in Fig. 2. Top panel, % peptide in super- a minor isotropic peak in the equimolar mixture of POPC and natant. Middle panel, % lipid in supernatant. Bottom panel, % choles- cholesterol. The slices for the sample of 3F with POPC alone terol in supernatant. (Fig. 12) generally had cross-peaks of positive sign (i.e. negative NOE), opposite to that with 3F-2. In addition, only the peak on motion in the presence of cholesterol decreasing the rate of the diagonal was observed in the aromatic region, indicating conformational change in the fluorescent probe. The effect of that the aromatic groups are less stacked in 3F than in 3F-2, the peptide is smaller and tends to increase with higher con- as might be anticipated on the basis of the larger cluster of centrations of probe, suggesting that there are both inter- and aromatic residues seen in the helical wheel projection of 3F-2 intra-molecular formation of excimers. This ratio is insensitive (Fig. 1). The cross-peaks between the lipid and 3F are par- to the presence of 3F but is affected by 3F-2, indicating that ticularly weak in the presence of cholesterol (Fig. 13), suggest- 3F-2 has a greater effect on hydrocarbon packing and/or ing that this peptide is largely excluded from cholesterol-con- dynamics. taining membranes. In addition, the aromatic region of the H NOESY MAS NMR—Slices from the two-dimensional one-dimensional spectrum is particularly well resolved com- NOESY spectrum of 3F-2 in the presence of POPC, at a 1:10 pared with other cases. peptide to lipid ratio, are presented for the spectral region of Interaction of the peptide with lipid can also be assessed by the aromatic side chain resonances using 50- or 300-ms mixing monitoring the changes in the chemical shifts of the lipid res- times (Fig. 10). The P NMR powder pattern demonstrated onances on introduction of the peptide (Table III). These that the major fraction of the lipid was in a bilayer arrange- changes in chemical shifts may arise from ring current effects ment, although there was a significant isotropic component in as well as from changes in the polarity of the environment. In the spectra of both peptides in the presence of POPC. There are the case of H MAS/NMR, only changes in the spectrum of the cross-peaks between the aromatic resonance and the protons phospholipid can be assessed, because resonances are not ob- from the lipid, particularly those of the CH protons, indicating served from protons of cholesterol in these lipid mixtures (13), insertion of the peptide into pure POPC membranes. The sign and the lower concentration of peptide makes it difficult to of the nuclear Overhauser enhancement with 50-ms mixing discern its resonances. The addition of 3F-2 or 3F to either time is negative, corresponding to rapid molecular motion, on POPC or POPC:cholesterol (1:1) results in a small change in H this time scale, between the peptide and lipid. In addition, for chemical shifts of several resonances. The change is insensitive several of the slices, peaks in the aromatic region, in addition to to the presence of cholesterol and does not indicate a preferen- 51410 Membrane Interactions of 3F Analogs FIG.7. Fluorescence emission spec- tra peptides with and without choles- terol. The left panel is for 3F , and the right panel is for 3F-2. Curve 1,15 M peptide in buffer. Curve 2,15 M peptide mixed with SOPC at a lipid to peptide ratio of 6; Curve 3,15 M peptide mixed with SOPC:cholesterol (1:1) at a lipid to peptide ratio of 6. Fluorescence intensi- ties normalized to make the emission maximum for peptide in buffer equal to 1.0. The spectra were acquired using an excitation wavelength of 280 nm at a tem- perature of 25 °C. FIG.9. Fluorescent properties of PC P at 1 mol % in LUVs of POPC or POPC-cholesterol (1:1) (lipid to peptide ratio  10). The peptide concentration was 24 M. The experiments were done at room temperature using an excitation wavelength of 344 nm. Excimer emis- sion intensity (I ) was read at 476 nm, and monomer emission intensity (I ) was read at 389 nm. contained in exchangeable plasma apolipoproteins, i.e. class A amphipathic helices (17). Although the position of the Trp residue in the two peptides is different, the nature and location of the other residues on the hydrophilic face of the helical conformation of these two peptides are identical, as is their FIG.8. The red edge excitation shift. The effect of excitation on total amino acid composition. The HPLC elution profile and the emission maximum of the intrinsic fluorescence of Trp. Upper panel, monolayer collapse pressure indicate that the two peptides 3F-2; lower panel,3F . f, peptide in PIPES buffer, pH 7.4 (30 M); , peptide in 1 mM SOPC with 15 mol % peptide; , peptide in 1 mM have similar hydrophobicities, but the 3F-2 is somewhat less SOPC:cholesterol (1:1) with 15 mol % peptide. The fluorescence was hydrophobic (5). Both peptides can rapidly solubilize 1-palmi- measured at 25 °C. toyl-2-oleoyl phosphatidylcholine at an equimolar ratio of pep- tide and lipid (5). The CD spectra of the two peptides are tial location of the peptide in the bilayer. Because cholesterol similar, and we show in this work that the secondary structure resonances are not observed in H MAS/NMR, we also meas- 13 is independent of the presence of cholesterol (Fig. 3). We also ured C MAS/NMR. The changes in chemical shift are gener- show that both peptides undergo a loss of secondary structure ally small, and there is no large difference between 3F-2 and 14 on heating (Fig. 4). However, the thermal denaturation of 3F-2 3F . In mixtures containing cholesterol the changes in chem- is more prominent in DSC scans of 3F-2 than of 3F (Fig. 2). ical shifts are comparable for POPC and for cholesterol (not Because a similar amount of helicity is lost upon heating of the shown). two peptides, we suggest that the transition of 3F-2 is more DISCUSSION cooperative because this peptide has a higher degree of oli- Using in vitro assays expected to correlate with protection gomerization on the membrane surface compared with 3F . against atherosclerosis, 3F-2 and 3F exhibit quite different We have shown that another biologically potent class A hel- potency (5); however, the differences in their biophysical prop- ical peptide, 4F, promoted the formation of cholesterol-rich erties have been found to be relatively small (this work and Ref. domains by preferentially interacting with the phospholipid 5). Both peptides are class A amphipathic helices and therefore component of cholesterol/SOPC mixtures (7). This finding pro- can fold into a helical structure resembling amphipathic helices vided an interesting contrast with the peptide LWYIK that Membrane Interactions of 3F Analogs 51411 FIG. 10. One-dimensional slices from the MAS H NOESY spectrum at the chemical shifts of the aromatic protons of a sample of POPC containing 10 mol % 3F-2. The mixing times were 50 ms (left panel) and 300 ms (right panel). The top spectra are conventional one-dimensional proton spectra of the samples. The resonance assignments are indicated on the top spectrum. FIG. 11. One-dimensional slices from the MAS H NOESY spectrum at the chemical shifts of the aromatic protons of a sample of POPC:cholesterol (1:1) containing 10 mol % 3F-2. The mixing times were 50 ms (left panel) and 300 ms (right panel). The top spectra are conventional one-dimensional proton spectra of the samples. The resonance assignments are indicated on the top spectrum. preferentially interacted with cholesterol (16). Rearrangement than are required for their formation in the absence of peptide of cholesterol in membranes can result from preferential inter- (Fig. 2 and Table I). The peptide 3F also promotes the forma- action of proteins with cholesterol-rich domains as well as with tion of cholesterol crystals. However, 3F does not lower the cholesterol-depleted domains (18). These observations are enthalpy of the phase transition of SOPC in mixtures with likely to be relevant to the mechanism of formation of “rafts” in cholesterol (Table II), indicating that it is not interacting pref- biological membranes. The DSC shows that 3F-2 interacts pref- erentially with SOPC. 3F also does not greatly increase the erentially with the phospholipid in mixtures of cholesterol and enthalpy of SOPC by removing cholesterol, as we had shown for SOPC. This is indicated by the fact that this peptide is much LWYIK (16). We suggest that a contributing factor to the pro- more potent in lowering the phase transition of SOPC than is motion of cholesterol segregation in the membrane is through 3F (Fig. 2 and Table II). 3F-2 also promotes the formation of an increase in the lateral pressure of the membrane “squeezing cholesterol crystals at mol fractions of cholesterol much lower out” cholesterol. The greater preference of 3F-2 for cholesterol- 51412 Membrane Interactions of 3F Analogs FIG. 12. One-dimensional slices from the MAS H NOESY spectrum at the chemical shifts of the aromatic protons of a sample of POPC containing 10 mol % 3F . The mixing times were 50 ms (left panel) and 300 ms (right panel). The top spectra are conventional one-dimensional proton spectra of the samples. The resonance assignments are indicated on the top spectrum. FIG. 13. One-dimensional slices from the MAS H NOESY spectrum at the chemical shifts of the aromatic protons of a sample of POPC:cholesterol (1:1) containing 10 mol % 3F . The mixing times were 50 ms (left panel) and 300 ms (right panel). The top spectra are conventional one-dimensional proton spectra of the samples. In the case of this figure the one-dimensional spectrum was more highly expanded to show the clearly resolved weak aromatic peaks. The resonance assignments are indicated on the top spectrum. depleted domains is also common to another biologically potent There is also a difference between the two peptides in how peptide of this series, 4F (7). This correlation is not seen with they interact with the membrane. The emission intensity from the less biologically active peptide 3F . the Trp of 3F-2 is increased more by lipid, especially the lipid Membrane Interactions of 3F Analogs 51413 TABLE III Changes in H chemical shifts of lipid resonances induced by 3F peptides Chemical shift difference With 3F-2 With 3F Resonance POPC/cholesterol POPC/cholesterol POPC POPC (1:1) (1:1) ppm Glycerol C2 0.04 0.04 0.05 0.02 Glycerol C3 0.03 0.03 0.02 0.02 Choline  0.03 0.03 0.03 0.03 Glycerol C1 0.03 0.04 0.03 0.03 Choline  0.03 0.03 0.02 0.05 Quaternary CH 0.03 0.03 0.02 0.02 CH CO 0.01 0.03 0 0.03 CH CCO 0.03 0.04 0.02 0.07 CH 0.03 0.02 0.02 0.01 Terminal CH 0.03 0.02 0.03 0.02 Chemical shift differences are in parts per million between that of lipid alone and in the presence of 10 mol% peptide. A positive charge corresponds to a shift in the resonance to a lower frequency caused by FIG. 14. Biological activity of a Class A amphipathic helix de- the peptide. pends on hydrophobic face-lipid acyl chain interaction. Top panel, a minimal effect on lipid acyl chain packing occurs in the wedge- 14 14 shaped molecule 3F . Bottom panel, the cylindrical shaped peptide, mixture containing cholesterol (Fig. 7), than is the case of 3F . 3F-2, causes greater acyl chain perturbations, facilitating the entry of The behavior of the Trp fluorescence of 3F-2 is qualitatively molecules such as water and lipid hydroperoxides into the hydrophobic similar to that of 4F (7), even though the Trp is in different milieu of the complex. positions for the two peptides. The similar behavior of the Trp fluorescence of two peptides, despite the different position of Trp in the helical wheel, suggests that 3F penetrates more The NMR parameters that we have monitored are not very deeply into the bilayer. The greater number of cross-peaks sensitive to the hydrocarbon packing or molecular motion in between protons of the aromatic residues of 3F-2 (Fig. 10) the interior of the membrane. We have therefore also studied indicates that its side chains are stacked in the presence of the properties of the fluorescent probe PC P that has been lipid. In the presence of cholesterol particularly, 3F-2 exhibits suggested to monitor changes in membrane motional proper- several strong, negative cross-peaks with several lipid protons, ties (12, 19). We do not wish to use the probe as the basis for a including those from the terminal CH group of the acyl chain. model of the interaction of the peptide with lipid but rather as The peaks of negative sign of the NOESY suggests increased a demonstration that the more active peptide, 3F-2, has a much molecular motion that could allow transient access to the pro- larger effect on the packing properties of the hydrocarbon re- tons in the center of the bilayer. This conclusion appears oppo- gion of the membrane, compared with the less active 3F . The site to that derived from red edge excitation shift, indicating a effects are consistent with the proposed model for the interac- restricted motion of the Trp of 3F-2. We suggest that this tion of these peptides with bilayers based on molecular shape difference is a consequence of the widely different time scale for (Fig. 14). In this model the 3F-2 peptide would introduce the two measurements. Fluorescence decay occurs in nanosec- greater destabilization of the bilayer as a consequence of its onds, whereas proton relaxation occurs in milliseconds to sec- cylindrical shape, short length, and aromatic side chains push- onds. Hence in the longer time scale of NMR, the relative ing the peptide axis up closer to the polar-nonpolar interface. position between the Trp and the lipid can change, but locally Accumulation of the probe in the bilayer defect would dilute the around the Trp the environment is rigid, and there is slow PC P and decrease intermolecular excimer formation and pos- reorientation of the surrounding dipoles. According to the sibly also disfavor the conformation required for pyrene dimer- model we proposed (Fig. 14), 3F-2 would, because of its cylin- ization in the monomer. This is also consistent with the greater drical shape, short length, depth of penetration, and aromatic anti-atherogenic properties of 3F-2, because decreased mem- side chains pushing the peptide axis up closer to the polar- brane order has been suggested to be associated with increased nonpolar interface, introduce a destabilization in the bilayer risk for cardiovascular disease (20). resulting in a decrease in the lipid order parameters. The In summary, given the marked differences in biological ac- 14 14 decreased penetration of 3F-2 compared with 3F is also indi- tivity between 3F-2 and 3F , the nature of the interaction of cated by the small shift in Trp emission wavelength caused by these peptides with SOPC with or without cholesterol is re- lipid (Fig. 7). Again there is a difference between peptide pen- markably similar. Nevertheless there are important differ- etration into the membrane as assessed by NOE effects and by ences between the two peptides that support our model of the fluorescence. It is possible that the greater bilayer disruption difference in peptide “shape” (5). Although 3F-2 is less hydro- caused by 3F-2 allows some penetration of water into the mem- phobic than 3F by the criteria of HPLC volume and mono- brane, resulting in a smaller effect on Trp emission, even layer exclusion pressure (5), 3F-2 has stronger NOESY cross- though the NOSEY spectra indicate a greater penetration of peaks with protons more in the center of the bilayer (Fig. 10). this peptide. In comparison, 3F appears to be largely ex- We suggest that this is a consequence of a greater disordering cluded from bilayers containing cholesterol. It shows only very of the bilayer caused by this peptide. 3F-2 also has two features weak cross-peaks between the aromatic resonances and the that are more in common with those of 4F (7). These are a lipid protons (Fig. 11). This is also in accord with the smaller preferential broadening of the chain melting transition in mix- change in the intensity of emission from the Trp residue in the tures of SOPC and cholesterol (Fig. 2) and a more cooperative presence of cholesterol. However, when 3F incorporates into unfolding transition of the peptide. The latter suggests oli- the bilayer, it may be able to pack with the acyl chains with less gomerization that may also contribute to a greater disruption disruption of the hydrocarbon portion of the bilayer, as sug- of the bilayer order by insertion of a larger peptide aggregate. gested in Fig. 14. The disordering of the lipid could allow for the transfer of 51414 Membrane Interactions of 3F Analogs (2004) J. Biol. Chem. 279, 26509–26517 oxidized lipids from the LDL surface to peptide-containing 6. Ou, Z., Ou, J., Ackerman, A. W., Oldham, K. T., and Pritchard, K. A., Jr. (2003) particles, thus rendering LDL less effective in inducing mono- Circulation 107, 1520–1524 7. Epand, R. M., Epand, R. F., Sayer, B. G., Melacini, G., Palgulachari, M. N., cyte chemotaxis, an important step in the initiation Segrest, J. P., and Anantharamaiah, G. M. (2004) Biochemistry 43, of atherogenesis. 5073–5083 8. Datta, G., Chaddha, M., Hama, S., Navab, M., Fogelman, A. M., Garber, D. W., Acknowledgment—We are grateful to Martin Jones for help with Mishra, V. K., Epand, R. M., Epand, R. F., Lund-Katz, S., Phillips, M. C., the figures. Segrest, J. P., and Anantharamaiah, G. M. (2001) J. Lipid Res. 42, 1096–1104 REFERENCES 9. Ames, B. N. (1966) Methods Enzymol. 8, 115–118 10. Zlatkis, A., Zak, B., and Boyle, A. J. (1953) J. Lab. Clin. Med. 41, 486–492 1. Navab, M., Anantharamaiah, G. M., Reddy, S. T., Van Lenten, B. 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Published: Dec 1, 2004

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