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• W2107887632 abstract "Human apolipoprotein E (apoE) mediates high affinity binding to the low density lipoprotein receptor when present on a lipidated complex. In the absence of lipid, however, apoE does not bind the receptor. Whereas the x-ray structure of lipid-free apoE3 N-terminal (NT) domain is known, the structural organization of its lipid-associated, receptor-active conformation is poorly understood. To study the organization of apoE amphipathic α-helices in a lipid-associated state, single tryptophan-containing apoE3 variants were employed in fluorescence quenching studies. The relative positions of the Trp residues with respect to the phospholipid component of apoE/lipid particles were established from the degree of quenching by phospholipids bearing nitroxide groups at various positions along their fatty acyl chains. Four apoE3-NT variants bearing Trp reporter groups at positions 141, 148, 155, or 162 within helix 4 and two apoE3 variants containing single Trp at positions 257 or 264 in the C-terminal (CT) domain, were reconstituted into phospholipid-containing discoidal complexes. Parallax analysis revealed that each engineered Trp residue in helix 4 of apoE3-NT, as well as those in the CT domain of apoE, localized ∼5 Å from the center of the bilayer. Circular dichroism studies revealed that lipid association induces additional helix formation in apoE. Protease protection assays suggest the flexible loop segment between the NT and CT domains may transition from unstructured to helix upon lipid association. Taken together, these data support a model wherein the α-helices in the receptor-binding region and the CT domain of apoE align perpendicular to the fatty acyl chains of the phospholipid bilayer. In this alignment, the residues of helix 4 are arrayed in a positively charged, curved helical segment for optimal receptor interaction. Human apolipoprotein E (apoE) mediates high affinity binding to the low density lipoprotein receptor when present on a lipidated complex. In the absence of lipid, however, apoE does not bind the receptor. Whereas the x-ray structure of lipid-free apoE3 N-terminal (NT) domain is known, the structural organization of its lipid-associated, receptor-active conformation is poorly understood. To study the organization of apoE amphipathic α-helices in a lipid-associated state, single tryptophan-containing apoE3 variants were employed in fluorescence quenching studies. The relative positions of the Trp residues with respect to the phospholipid component of apoE/lipid particles were established from the degree of quenching by phospholipids bearing nitroxide groups at various positions along their fatty acyl chains. Four apoE3-NT variants bearing Trp reporter groups at positions 141, 148, 155, or 162 within helix 4 and two apoE3 variants containing single Trp at positions 257 or 264 in the C-terminal (CT) domain, were reconstituted into phospholipid-containing discoidal complexes. Parallax analysis revealed that each engineered Trp residue in helix 4 of apoE3-NT, as well as those in the CT domain of apoE, localized ∼5 Å from the center of the bilayer. Circular dichroism studies revealed that lipid association induces additional helix formation in apoE. Protease protection assays suggest the flexible loop segment between the NT and CT domains may transition from unstructured to helix upon lipid association. Taken together, these data support a model wherein the α-helices in the receptor-binding region and the CT domain of apoE align perpendicular to the fatty acyl chains of the phospholipid bilayer. In this alignment, the residues of helix 4 are arrayed in a positively charged, curved helical segment for optimal receptor interaction. Apolipoprotein E (ApoE) is a key regulator of plasma cholesterol homeostasis. Its interactions with the low density lipoprotein receptor (LDLR) 1The abbreviations used are: LDLR, low density lipoprotein receptor; apoE, apolipoprotein E; HDL, high density lipoprotein; rHDL, reconstituted HDL; DMPC, dimyristoylphosphatidylcholine; PL, phospholipid; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; PSPC, 1-palmitoyl-2-stearoyl-phosphatidylcholine; λmax, wavelength of maximum fluorescence. 1The abbreviations used are: LDLR, low density lipoprotein receptor; apoE, apolipoprotein E; HDL, high density lipoprotein; rHDL, reconstituted HDL; DMPC, dimyristoylphosphatidylcholine; PL, phospholipid; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; PSPC, 1-palmitoyl-2-stearoyl-phosphatidylcholine; λmax, wavelength of maximum fluorescence. family and cell surface heparan sulfate proteoglycans (1Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Google Scholar, 2Mahley R.W. Huang Y. Curr. Opin. Lipidol. 1999; 10: 207-217Google Scholar, 3Brown M.S. Goldstein J.L. Science. 1986; 232: 34-47Google Scholar) are a critical step for the cellular uptake of apoE-containing lipoproteins. Transgenic mice overexpressing apoE manifest decreased plasma cholesterol levels on chow diet and a marked resistance to hypercholesterolemia on a high cholesterol/fat diet (4Shimano H. Yamada N. Katsuki M. Yamamoto K. Gotoda T. Harada K. Shimada M. Yazaki Y. J. Clin. Investig. 1992; 90: 2084-2091Google Scholar). On the other hand, apoE-deficient subjects display features of type III hyperlipoproteinemia (5Schaefer E.J. Gregg R.E. Ghiselli G. Forte T.M. Ordovas J.M. Zech L.A. Brewer Jr., H.B. J. Clin. Investig. 1986; 78: 1206-1219Google Scholar) and apoE null mice exhibit massive accumulation of remnant lipoproteins (6Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Google Scholar), documenting the direct relevance of apoE in lipoprotein metabolism. However, structural features of lipid-associated apoE responsible for its receptor interaction properties are not understood at the molecular level.ApoE comprises two independently folded structural and functional domains that are linked by a protease-sensitive loop segment. The globular 22 kDa N-terminal (NT) domain houses the LDLR recognition site, whereas the 10 kDa C-terminal (CT) domain bears high affinity lipoprotein-binding and self-association sites (7Aggerbeck L.P. Wetterau J.R. Weisgraber K.H. Wu C.S. Lindgren F.T. J. Biol. Chem. 1988; 263: 6249-6258Google Scholar, 8Wetterau J.R. Aggerbeck L.P. Rall Jr., S.C. Weisgraber K.H. J. Biol. Chem. 1988; 263: 6240-6248Google Scholar). The NT domain is composed of four elongated amphipathic α-helices organized as an up-and-down helix bundle in the absence of lipid (9Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Google Scholar, 10Segelke B.W. Forstner M. Knapp M. Trakhanov S.D. Parkin S. Newhouse Y.M. Bellamy H.D. Weisgraber K.H. Rupp B. Protein Sci. 2000; 9: 886-897Google Scholar). Helix 4 of the NT domain (residues 131-164) harbors key residues required for interaction with lipoprotein receptors (1Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Google Scholar). However, lipid association is a requirement for apoE (full-length or NT domain) to display receptor recognition capability, with apoE bound to model lipid particles displaying receptor binding ability comparable with that of native apoE-containing lipoproteins (11Innerarity T.L. Pitas R.E. Mahley R.W. J. Biol. Chem. 1979; 254: 4186-4190Google Scholar). Upon interaction with lipid it has been proposed that the NT domain helix bundle “opens” to expose the hydrophobic faces of its amphipathic helices to potential lipid surface binding sites, thereby achieving a receptor-active conformation (1Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Google Scholar, 12Fisher C.A. Ryan R.O. J. Lipid Res. 1999; 40: 93-99Google Scholar). Monolayer surface balance studies at the air/water interface provide evidence that the NT domain occupies a larger surface area than can be accounted for by its globular 4-helix bundle conformation (13Weisgraber K.H. Lund-Katz S. Phillips M.C. Miller N.E. Tall A.R. High Density Lipoproteins and Atherosclerosis. III. Elsevier, Amsterdam1992: 175-181Google Scholar), consistent with adoption of an “open” conformation. Subsequent fluorescence energy transfer analysis revealed that helical segments in the NT domain realign as a function of the transition from lipid-free helix bundle to lipid-bound state (12Fisher C.A. Ryan R.O. J. Lipid Res. 1999; 40: 93-99Google Scholar, 14Fisher C.A. Narayanaswami V. Ryan R.O. J. Biol. Chem. 2000; 275: 33601-33606Google Scholar). Attenuated total reflectance Fourier-transformed infrared spectroscopic studies (15Raussens V. Fisher C.A. Goormaghtigh E. Ryan R.O. Ruysschaert J.M. J. Biol. Chem. 1998; 273: 25825-25830Google Scholar) indicate that apoE-NT α-helices orient perpendicular to phospholipid (PL) acyl chains in discoidal complexes, although another report indicates a parallel orientation (16De Pauw M. Vanloo B. Weisgraber K. Rosseneu M. Biochemistry. 1995; 34: 10953-10966Google Scholar). Recent NMR spectroscopy studies provided evidence for increased solvent exposure of positively charged amino acid side chains in the receptor-binding region upon interaction of the NT domain with lipid (17Lund-Katz S. Zaiou M. Wehrli S. Dhanasekaran P. Baldwin F. Weisgraber K.H. Phillips M.C. J. Biol. Chem. 2000; 275: 34459-34464Google Scholar).With regard to the CT domain, the precise molecular features responsible for high affinity lipid binding are not known, although it is believed that CT domain interaction with lipids is mediated by putative amphipathic α-helices (1Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Google Scholar). Using deletion mutagenesis, Westerlund and Weisgraber (18Westerlund J.A. Weisgraber K.H. J. Biol. Chem. 1993; 268: 15745-15750Google Scholar) demonstrated that residues 267-299 contribute to the lipoprotein binding properties of apoE. In other studies, Dong et al. (19Dong L.M. Wilson C. Wardell M.R. Simmons T. Mahley R.W. Weisgraber K.H. Agard D.A. J. Biol. Chem. 1994; 269: 22358-22365Google Scholar) reported that residues 260-272 are important for complete lipoprotein association.To address questions relating to the alignment and orientation of the receptor-binding region of the NT domain and lipid-binding sites in the CT domain of lipid-associated apoE, we examined the relative ability of spatially defined nitroxide-labeled PL to quench the fluorescence of a panel of unique single Trp apoE3 variants in reconstituted high density lipoproteins (rHDL). It was determined that the engineered Trp residues in helix 4 align uniformly, close to the center of the PL bilayer, thereby presenting the receptor-binding site as a positively charged, curved helical segment analogous to that displayed on spherical lipoprotein particles.EXPERIMENTAL PROCEDURESMaterials—1-Palmitoyl-2-oleoylphosphatidylcholine (POPC) and the nitroxide spin probes 1-palmitoyl-2-stearoyl-(X-DOXYL)-sn-glycero-3-phosphocholine (where X = 5, 7, 10, 12, or 16) and dimyristoylphosphatidylcholine (DMPC) were purchased from Avanti Polar Lipids (Birmingham, AL). The single Trp synthetic peptides K2WL9AL9K2A and K2GL9WL9K2A were a generous gift of Dr. Erwin London at the State University of New York at Stony Brook. Human thrombin was obtained from Hematologic Technologies Inc. All other reagents were analytical grade.Site-directed Mutagenesis, Protein Expression, and Purification—Single Trp mutants were introduced in a Trp null apoE3-NT (residues 1-183) variant by site directed-mutagenesis and expressed in Escherichia coli as described (14Fisher C.A. Narayanaswami V. Ryan R.O. J. Biol. Chem. 2000; 275: 33601-33606Google Scholar, 20Fisher C.A. Wang J. Francis G.A. Sykes B.D. Kay C.M. Ryan R.O. Biochem. Cell Biol. 1997; 75: 45-53Google Scholar). Full-length apoE3 bearing single Trp residues at position 257 or 264 were constructed using the QuikChange® mutagenesis protocol (Stratagene, La Jolla, CA) in a strategy similar to that described elsewhere (14Fisher C.A. Narayanaswami V. Ryan R.O. J. Biol. Chem. 2000; 275: 33601-33606Google Scholar). All constructs were verified by restriction analysis and double-stranded DNA sequencing in both directions. Single Trp-containing apoE3 variants were expressed and purified as thioredoxin fusion proteins in E. coli upon subcloning into a pET32a(+) vector (17Lund-Katz S. Zaiou M. Wehrli S. Dhanasekaran P. Baldwin F. Weisgraber K.H. Phillips M.C. J. Biol. Chem. 2000; 275: 34459-34464Google Scholar, 21Morrow J.A. Arnold K.S. Weisgraber K.H. Protein Expression Purif. 1999; 16: 224-230Google Scholar) following manipulations to optimize codon usage for bacterial expression (22Vogel T. Weisgraber K.H. Zeevi M.I. Ben Artzi H. Levanon A.Z. Rall Jr., S.C. Innerarity T.L. Hui D.Y. Taylor J.M. Kanner D. Yavin Z. Amit B. Aviv H. Gorecki M. Mahely R.W. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8696-8700Google Scholar). Intact apoE preparations without the GST fusion protein were at least 95% pure as assessed by SDS-PAGE.Preparation and Characterization of rHDL—ApoE3 variants (2-3 mg/ml) were dissolved in 100 mm ammonium bicarbonate, pH 8.0, containing 6 m guanidine HCl and 5 μl of β-mercaptoethanol/mg of protein. The solutions were dialyzed against 100 mm ammonium bicarbonate, pH 8.0. rHDL were prepared as described previously (23Maiorano J.N. Davidson W.S. J. Biol. Chem. 2000; 275: 17374-17380Google Scholar) using POPC, a specified apoE3 variant, a given DOXYL-PL, and sodium cholate, except that the lipids were subjected to bath sonication for 30 min prior to addition of sodium cholate. The DOXYL moieties were located at positions C-5, C-7, C-10, C-12, or C-16 along the stearoyl chain of POPC at the sn-2 position. Initial PL:DOXYL-PL:apoE molar ratios were 49:9:1. For each apoE3 variant, particles were reconstituted with and without DOXYL-PL to characterize the Trp fluorescence contribution in the presence and the absence of quencher, respectively. In rHDL lacking DOXYL-PL, the corresponding PL mass was replaced by POPC to maintain uniformity in particle lipid to protein ratio. The rHDL were isolated by gel filtration chromatography on a Superdex 200 gel filtration column (24Davidson W.S. Rodrigueza W.V. Lund-Katz S. Johnson W.J. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1995; 270: 17106-17113Google Scholar) to remove unbound protein and/or vesicular structures. Particle hydrodynamic properties were assessed by gradient native polyacrylamide electrophoresis and/or Superdex 200 gel filtration chromatography (24Davidson W.S. Rodrigueza W.V. Lund-Katz S. Johnson W.J. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1995; 270: 17106-17113Google Scholar).Fluorescence Spectroscopy—Fluorescence measurements were performed on a Photon Technology International Quantamaster spectrometer in photon counting mode. For each variant, an aliquot of the rHDL samples, with or without quenchers, was diluted in buffer. Fluorescence emission spectra were recorded from 303 to 375 nm at an excitation wavelength of 295 nm in order to minimize the contribution of tyrosine fluorescence in apoE. Emission and excitation band-passes were 3.0 nm. Fluorescence analysis was carried out at 25 °C in a semi-microquartz cuvette. The spectra were corrected for background fluorescence of buffer alone. The spectra were not corrected for the spectral characteristics of the emission and excitation monochromators.Trp Depth Calculations—The parallax method for determining the depth of penetration of a fluorophore in a lipid bilayer was derived by Chattopadhyay and London (25Chattopadhyay A. London E. Biochemistry. 1987; 26: 39-45Google Scholar) from conventional relationships of static quenchers to randomly distributed fluorophores. The differences in fluorescence intensities of the fluorophore in the presence of known concentrations of nitroxide quencher groups at known locations in a PL acyl chain are used to calculate the depth of the fluorophore relative to the quenchers (see Ref. 23Maiorano J.N. Davidson W.S. J. Biol. Chem. 2000; 275: 17374-17380Google Scholar for a detailed discussion of the method theory). The distance of the Trp from the center of the bilayer (Zcf) is given by Zcf=Lcs+[-ln(Fs/Fd)/πC-Lds2]/2Lds(Eq. 1) where Fs is the fluorescence intensity in the presence of the shallow quencher, Fd is the same for the deep quencher, Lcs is the distance from the center of the bilayer to the shallow quencher, Lds is the distance between the shallow and deep quenchers, and C is the concentration of the quencher molecules per Å2. Equation 1 is valid when nitroxide groups that are shallow in the membrane quench the Trp residue. When the Trp is buried deep within the membrane, however, it is subjected to quenching from groups in the opposite leaflet the lipid bilayer. For this situation, a second relationship is required to account for trans-bilayer quenching, Zcf=Lcd-[ln((Fs/Fo)2/(Fd/Fo))/πC)-2Lds2+4Lcd2)/4(Lds+Lcd)](Eq. 2) where F0 is the fluorescence intensity in the absence of quencher, Lcd is the distance from the center of the bilayer to the deep quencher, and Lds is the distance between the shallow and deep quenchers. Equation 2 was used in this study whenever a Trp residue was determined to be <6 Å from the center of the membrane by Equation 1.Circular Dichroism Spectroscopy—Circular dichroism (CD) spectra were collected on a Jasco J600 or J720 spectropolarimeter equipped with a temperature-controlling device and interfaced with a computer. The molar ellipticity ([θ]) in degrees cm2 dmol-1 at 222 or 208 nm was calculated from the equation [θ] = (MRW·θ)/(10d·c), where MRW is the mean residue weight of the apoE CT domain calculated to be 114.31, θ is the measured ellipticity in degrees at 222 or 208 nm, d is the cuvette path length in cm, and c is the protein concentration in g/ml. The α-helical content of each rHDL preparation was derived from the molar ellipticity at 222 nm by established procedures (26Lund-Katz S. Weisgraber K.H. Mahley R.W. Phillips M.C. J. Biol. Chem. 1993; 268: 23008-23015Google Scholar, 27Sparks D.L. Phillips M.C. Lund-Katz S. J. Biol. Chem. 1992; 267: 25830-25838Google Scholar). In other analyses, lipid-free apoE3 was incubated overnight in the absence or in the presence of trifluoroethanol (50% v/v) prior to CD analysis. In addition, spectra of apoE3-DMPC complexes were obtained (28Narayanaswami V. Szeto S.S. Ryan R.O. J. Biol. Chem. 2001; 276: 37853-37860Google Scholar).Thrombin Digestion—The effect of lipid association on accessibility to thrombin digestion was examined by treating 20 μg of apoE3 in a lipid-free or DMPC-bound state (14Fisher C.A. Narayanaswami V. Ryan R.O. J. Biol. Chem. 2000; 275: 33601-33606Google Scholar) in 20 mm Tris-HCl, pH 8.5, 150 mm NaCl, 2.5 mm CaCl2 with 1 milliunit of thrombin for 3 h at 37 °C. The samples were analyzed on a 4-20% acrylamide gradient by SDS-PAGE with untreated lipid-free or -bound apoE3 as control.Analytical Procedures—Protein concentrations were determined by the Lowry procedure (29Lowry O.H. Rosenbrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Google Scholar) or by A280 measurements using an extinction coefficient of 1.34 for 1 mg of full-length apoE3 protein/ml or 1.32 for 1 mg of apoE3 NT domain protein/ml. PL content was determined by phosphorus analysis (30Sokoloff L. Rothblat G.H. Proc. Soc. Exp. Biol. Med. 1974; 146: 1166-1172Google Scholar).RESULTSExperimental Strategy—Receptor binding analyses of genetically engineered and naturally occurring variants of apoE have demonstrated that residues in the region of residues 134-150 of apoE3 are indispensable for receptor binding activity (1Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Google Scholar). High resolution structural information on lipid-free human apoE3-(1-191) reveals that helix 4 spans residues 131-164 (9Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Google Scholar), thereby encompassing the entire receptor-binding element. Using a variety of biophysical approaches, it has been shown that the NT domain helix bundle undergoes a conformational change upon interaction with lipid (see Ref. 31Narayanaswami V. Ryan R.O. Biochim. Biophys. Acta. 2000; 1483: 15-36Google Scholar for a review). Whereas the precise structural alteration that accompanies lipid association of the NT domain is not known, this conformational adaptation confers the protein with LDLR recognition properties (32Innerarity T.L. Friedlander E.J. Rall Jr., S.C. Weisgraber K.H. Mahley R.W. J. Biol. Chem. 1983; 258: 12341-12347Google Scholar). Because the lipid-bound apoE3-NT mimics full-length apoE3 in terms of receptor binding activity, we used this domain in studies designed to characterize the orientation and positioning of helix 4 in lipid-associated apoE3-NT. Isolated recombinant apoE3-NT domain was reconstituted with PL to generate a homogeneous population of discoidal lipid particles that are known to be LDLR-active (32Innerarity T.L. Friedlander E.J. Rall Jr., S.C. Weisgraber K.H. Mahley R.W. J. Biol. Chem. 1983; 258: 12341-12347Google Scholar). To evaluate the disposition of the receptor-interaction site with respect to the lipid surface, a series of apoE3-NT domain variants possessing a single Trp residue positioned along the length of helix 4 (at positions 141, 148, 155, or 162; see Fig. 1) were subjected to fluorescence spectroscopy. By examining the relative ability of a series of DOXYL-PL (with the nitroxide moiety located at different positions along the fatty acyl chain) to quench apoE3-NT Trp fluorescence, the relative position of the Trp probes with respect to PL bilayer can be deduced. For example, if helix 4 retains its general organization and aligns with its helical axis parallel to the PL acyl chains, large differences in nitroxide quenching behavior would be anticipated for the different single Trp variants positioned at every second turn of the helix. On the other hand, if helix 4 aligns perpendicular to the PL acyl chains, the extent of nitroxide quenching will be similar for each of the single Trp variants.Characterization of apoE3 Variant Lipid Particles—Reconstituted lipid particles composed of POPC, a given DOXYL-PL, and a unique single Trp apoE3 variant, were found to be of uniform size as judged by native gradient PAGE analysis. In each case the particles displayed an average diameter of 115 ± 10 Å, consistent with findings reported for wild type apoE3-NT domain (20Fisher C.A. Wang J. Francis G.A. Sykes B.D. Kay C.M. Ryan R.O. Biochem. Cell Biol. 1997; 75: 45-53Google Scholar). No differences were observed in the physical characteristics of complexes prepared with POPC alone versus POPC plus DOXYL-PL. A similar lack of a structural perturbation from the DOXYL-PL was previously noted for particles made with apoA-I (23Maiorano J.N. Davidson W.S. J. Biol. Chem. 2000; 275: 17374-17380Google Scholar, 33Panagotopulos S.E. Horace E.M. Maiorano J.N. Davidson W.S. J. Biol. Chem. 2001; Google Scholar). Taken together, these data indicate that the apoE3 variants employed in this study retained their lipid binding ability and that incorporation of DOXYL-PL in the reconstituted particles did not significantly alter their size or hydrodynamic properties. Furthermore, far UV circular dichroism analysis of a subset of the disc particles employed in this study consistently yielded 70-75% α-helix content.Trp Fluorescence Studies—Prior to analysis of the Trp quenching patterns in rHDL containing the different single Trp apoE3 variants, control experiments were performed to validate the quenching protocol and to ensure that the nitroxide moieties quench at the expected depth under the conditions employed to reconstitute apoE3 into lipid particles. Two hydrophobic α-helical peptides that possess a single Trp residue at different points along the length of the helix were employed. Ren et al. (34Ren J. Lew S. Wang Z. London E. Biochemistry. 1997; 36: 10213-10220Google Scholar) have previously demonstrated that these peptides traverse the membrane of small unilamellar vesicles and orient parallel to the acyl chains. Peptide 1 (K2WL9AL9K2A) contains a membrane-spanning sequence composed primarily of repeating leucines flanked by pairs of lysine residues. The Trp in peptide 1 is located near one end of the helix. Peptide 2 (K2GL9WL9K2A) is similar in design but contains a Trp at the center of the peptide sequence. Hence, when inserted into a membrane the Trp in peptide 1 is expected to locate close to the polar/apolar interface, as depicted in Fig. 2A, whereas peptide 2 is expected to be located near the center of the bilayer. Reconstitution of peptides 1 and 2 into vesicles containing specific DOXYL-PL and fluorescence analysis revealed that the Trp in K2WL9AL9K2A was maximally quenched when the DOXYL moiety was located at position C-5, 12 Å from the bilayer center (Fig. 2B). The extent of quenching observed decreased as the DOXYL moiety was positioned further down the acyl chain. Peptide 2 exhibited the opposite quenching pattern, wherein Trp fluorescence was minimally quenched when the DOXYL moiety was located at C-5 and was maximally quenched by C-16 DOXYL-PL (2 Å from the bilayer center). The calculated depths of these Trp residues were 15.4 ± 2.2 Å for peptide 1 and 3.0 ± 2.6 Å from the bilayer center for peptide 2.Fig. 2Nitroxide lipid quenching of single Trp-containing, transmembrane peptides in small unilamellar vesicles. A, schematic representation of the positioning of PL DOXYL moieties (black circles) with respect to a model transmembrane helix. The situation for both the shallowest (C-5, left) and deepest (C-12, right) DOXYL moieties are shown. The asterisk indicates the shallow Trp in peptide 1 (K2WL9AL9K2A). B, small unilamellar vesicles containing peptide 1 (K2WL9AL9K2A (filled circles)) or peptide 2 (K2GL9WL9K2A (open circles)) and X-DOXYL-PL (where X = carbon position 2, 6, 8, 10, or 12 on the fatty acyl chain) were prepared as described under “Experimental Procedures.” The molar ratio of POPC:X-DOXYL-PL:peptide was 65:12:1. Emission spectra were recorded between 303 and 375 nm (excitation wavelength of 295 nm), and F/F0 was plotted as a function of the calculated distance of the various DOXYL groups on the fatty acyl chains from the center of the bilayer (34Ren J. Lew S. Wang Z. London E. Biochemistry. 1997; 36: 10213-10220Google Scholar).View Large Image Figure ViewerDownload (PPT)ApoE3 NT rHDL—The wavelength of maximal fluorescence emission intensity (λmax) for each single Trp apoE3 variant (excitation 295 nm) was determined in the absence of nitroxide labeled PL (Table I). In each case reconstitution of the single Trp variant apoE3 NT variant with POPC resulted in a λmax value of ∼330-332 nm. Considering that solvent-exposed Trp residues exhibit λmax values in the range of 350 nm, this result indicates that these Trp residues are not exposed to the aqueous environment and, therefore, are well suited for lipid-based quenching studies. We then determined the effect of specific DOXYL-PL on the fluorescence intensity of lipid associated single Trp apoE3-NT variants. Data are shown as a plot of F/F0 (where F and F0 represent fluorescence intensities in the presence and absence of quencher, respectively) versus DOXYL moiety distance from the PL bilayer center. In the case of Trp-141 apoE3-NT, the DOXYL-PL with the quencher located at position C-5 of the fatty acyl chain (12 Å from bilayer center), was the least effective (Fig. 3, top panel). As the DOXYL moiety was positioned further along the fatty acyl chain (i.e. deeper into the membrane), Trp-141 fluorescence was increasingly quenched, with maximal quenching observed with quencher located at the C-12 position and decreasing at C-16. ApoE3-NT variants with Trp at position 148 or 162 displayed a similar pattern of quenching. In the case of Trp-155 apoE3 NT domain, we confined our study to lipid particles possessing DOXYL moieties at positions C-5 and C-12 because of sample limitations. Despite this, we were able to calculate a reliable depth of penetration for this variant (see below). To determine the precise location of the Trp residues with respect to the bilayer, the distance of each Trp from the center of the bilayer was calculated using parallax analysis (25Chattopadhyay A. London E. Biochemistry. 1987; 26: 39-45Google Scholar). This method uses the quenching ratio from a pair of quenchers, one shallow and one deep in the membrane (see “Experimental Procedures”). PL with DOXYL moieties at C-5 and C-12 were chosen for the calculation for two reasons: (i) this pair provides information close to the bilayer surface and relatively close to the bilayer center, respectively, and (ii) detailed information for the quenching of the two control peptides (employed in this study) and 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled phospholipid with quenchers located at positions C-5 and C-12 is available (35Abrams F.S. London E. Biochemistry. 1993; 32: 10826-10831Google Scholar, 36Baratti J. Maroux S. Biochim. Biophys. Acta. 1976; 452: 488-496Google Scholar). Table I summarizes the fluorescence characteristics and quenching data for the rHDL particles of the apoE3 variants. Each of the four single Trp apoE3-NT variants exhibited a narrow range of depth of location, between 4.4 and 5.7 Å, indicating that positions 141, 148, 155, and 162 along the hydrophobic face of helix 4 localize to similar depths in the PL bilayer.Table IFluorescence characteristics and parallax analysis of single Trp apoE3 variants in rHDL particlesApoE3 variantλmaxaWavelength of maximum fluorescence.ZcfbDepth of penetration of the Trp residue measured from the bilayer center (0 Å); calculated from Equations 1 and 2 under “Experimental Procedures.”nmÅNT domainTrp-141330.0cBecause of poor recovery from the gel filtration column, these samples were measured in duplicat" @default.
• W2107887632 created "2016-06-24" @default.
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• W2107887632 date "2004-04-01" @default.
• W2107887632 modified "2023-10-15" @default.
• W2107887632 title "Helix Orientation of the Functional Domains in Apolipoprotein E in Discoidal High Density Lipoprotein Particles" @default.
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