The Interaction of Peripheral Proteins and Membranes Studied with α-Lactalbumin and Phospholipid Bilayers of Various Compositions
2003; Elsevier BV; Volume: 278; Issue: 24 Linguagem: Inglês
10.1074/jbc.m211466200
ISSN1083-351X
AutoresArmelle Varnier Agasøster, Øyvind Halskau, Edvin Fuglebakk, Nils Åge Frøystein, Arturo Muga, Holm Holmsen, Aurora Martı́nez,
Tópico(s)Metabolism and Genetic Disorders
ResumoTo characterize the interaction of peripheral proteins and membranes at the molecular level, we studied the reversible association of bovine α-lactalbumin (BLA) with lipid bilayers composed of different molecular forms of phosphatidylserine or equimolar mixtures of these phosphatidylserine forms and egg yolk phosphatidylcholine. At pH 4.5, almost all BLA (>90%) associates to negatively charged small unilamellar vesicles. The conformational changes that binding to these bilayers induced on the protein were characterized by circular dichroism and fluorescence spectroscopy. Because binding of BLA to negatively charged vesicles is reverted by adjusting the pH back to >6.0, we also investigated the conformation of the membrane-bound protein by NMR-monitored H-D exchange of the backbone amide protons. The conformation adopted by BLA bound to these bilayers resembles a molten globule-like state but the negative ellipticity at 222 nm and the apparent α-helix content of the bound protein senses the changes in the physical properties of the membrane. Binding to bilayers in the gel state appears to correlate with an increased amount of α-helical structure and with a lower extent of integration into the membrane, corresponding to the adsorbed protein, while the opposite is found for BLA bound to vesicles in the liquid-crystalline phase, corresponding to the embedded conformation. A common feature for the membrane-bound conformations of BLA is that the amphipathic helix C (residues 86 to 99) is an important determinant for the adsorption and further integration of the protein into the membrane. To characterize the interaction of peripheral proteins and membranes at the molecular level, we studied the reversible association of bovine α-lactalbumin (BLA) with lipid bilayers composed of different molecular forms of phosphatidylserine or equimolar mixtures of these phosphatidylserine forms and egg yolk phosphatidylcholine. At pH 4.5, almost all BLA (>90%) associates to negatively charged small unilamellar vesicles. The conformational changes that binding to these bilayers induced on the protein were characterized by circular dichroism and fluorescence spectroscopy. Because binding of BLA to negatively charged vesicles is reverted by adjusting the pH back to >6.0, we also investigated the conformation of the membrane-bound protein by NMR-monitored H-D exchange of the backbone amide protons. The conformation adopted by BLA bound to these bilayers resembles a molten globule-like state but the negative ellipticity at 222 nm and the apparent α-helix content of the bound protein senses the changes in the physical properties of the membrane. Binding to bilayers in the gel state appears to correlate with an increased amount of α-helical structure and with a lower extent of integration into the membrane, corresponding to the adsorbed protein, while the opposite is found for BLA bound to vesicles in the liquid-crystalline phase, corresponding to the embedded conformation. A common feature for the membrane-bound conformations of BLA is that the amphipathic helix C (residues 86 to 99) is an important determinant for the adsorption and further integration of the protein into the membrane. As a part of their functions, some intracellular proteins can reversibly translocate between the cytosol and membrane surfaces, leading to a change in conformation and a consequent variation in activity (1Johnson J.E. Cornell R.B. Mol. Membr. Biol. 1999; 16: 217-235Crossref PubMed Scopus (240) Google Scholar, 2Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar, 3Yang W. Boggs K.P. Jackowski S. J. Biol. Chem. 1995; 270: 23951-23957Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 4Cornell R.B. Northwood I.C. Trends Biochem. Sci. 2000; 25: 441-447Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Similarly, extracellular proteins such as apolipoproteins can alternatively exist free in plasma or bound to lipoprotein lipids, in which case a new conformation is induced (5Fisher C.A. Ryan R.O. J. Lipid Res. 1999; 40: 93-99Abstract Full Text Full Text PDF PubMed Google Scholar, 6Sahoo D. Narayanaswami V. Kay C.M. Ryan R.O. J. Biol. Chem. 1998; 273: 1403-1408Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 7Soulages J.L. Arrese E.L. J. Biol. Chem. 2000; 275: 17501-17509Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 8Wang S.X. Sun Y.T. Sui S.F. Biochem. J. 2000; 348: 103-106Crossref PubMed Scopus (42) Google Scholar). The membrane-bound form of these amphitropic proteins is adsorbed or partially embedded in the lipidic surfaces. Secreted soluble toxins may be inserted through both leaflets of the membrane and in vitro studies have shown a transient membrane-triggered shift of their conformation that is necessary for their insertion in the membrane (9Lakey J.H. Parker M.W. Gonzalez-Mañas J.M. Duche D. Vriend G. Baty D. Pattus F. Eur. J. Biochem. 1994; 220: 155-163Crossref PubMed Scopus (26) Google Scholar, 10Caaveiro J.M. Echabe I. Gutierrez-Aguirre I. Nieva J.L. Arrondo J.L. Gonzalez-Manas J.M. Biophys. J. 2001; 80: 1343-1353Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Among the different factors that modulate the association of proteins with the membrane, the lipid composition seems to be determinant (1Johnson J.E. Cornell R.B. Mol. Membr. Biol. 1999; 16: 217-235Crossref PubMed Scopus (240) Google Scholar, 2Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar, 11Dahim M. Brockman H. Biochemistry. 1998; 37: 8369-8377Crossref PubMed Scopus (35) Google Scholar, 12Killian J.A. von Heijne G. Trends Biochem. Sci. 2000; 25: 429-434Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). The composition of the lipid bilayer can also act specifically on the conformation of proteins adsorbed or inserted in the membrane (13Kirsch T. Nah H.D. Demuth D.R. Harrison G. Golub E.E. Adams S.L. Pacifici M. Biochemistry. 1997; 36: 3359-3367Crossref PubMed Scopus (86) Google Scholar, 14Braschi S. Neville T.A. Vohl M.C. Sparks D.L. J. Lipid Res. 1999; 40: 522-532Abstract Full Text Full Text PDF PubMed Google Scholar, 15Baenziger J.E. Morris M.L. Darsaut T.E. Ryan S.E. J. Biol. Chem. 2000; 275: 777-784Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 16Gadd M.E. Biltonen R.L. Biochemistry. 2000; 39: 9623-9631Crossref PubMed Scopus (35) Google Scholar). The soluble, calcium-binding milk protein bovine α-lactalbumin (BLA) 1The abbreviations used are: BLA, bovine α-lactalbumin; DOPG, 1,2-dioleoyl-sn-glycero-3-(phospho-1-glycerol); DSPS, 1,2-distearoyl-sn-glycero-3-(phospho-l-serine); EYPC, egg yolk phosphatidylcholine; LUV, large unilamellar vesicles; SOPS, 1-stearoyl-2-oleoyl-sn-glycero-3-(phospho-l-serine); POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-l-serine); PS, phosphatidylserine; SUV, small unilamellar vesicles. is a component of the lactose synthase complex. BLA binds to galactosyltransferase, promoting glucose binding and facilitating the synthesis of lactose in the lactating mammary gland (17Schanbacher F.L. Ebner K.E. Biochim. Biophys. Acta. 1971; 229: 226-232Crossref PubMed Scopus (7) Google Scholar, 18Permyakov E.A. Berliner L.J. FEBS Lett. 2000; 473: 269-274Crossref PubMed Scopus (424) Google Scholar). BLA can also reversibly associate with lipid membranes under specific conditions. Thus, it has been shown that at pH 4.5, calcium-containing BLA binds to negatively charged liposomes and that the binding is reverted by adjusting the pH back to >6.0 (19Bañuelos S. Muga A. J. Biol. Chem. 1995; 270: 29910-29915Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The protein only adsorbs to vesicles made of saturated lipids without disrupting the permeability barrier of the bilayer, whereas it adopts a partial embedded (inserted) state upon binding to vesicles of unsaturated lipids (in the liquid-crystalline phase) able to disrupt the bilayer (20Bañuelos S. Muga A. FEBS Lett. 1996; 386: 21-25Crossref PubMed Scopus (28) Google Scholar). Our recent NMR studies have lead to a mechanism for the partial insertion of BLA into negatively charged membranes that includes initial protonation of acidic side chains at the membrane interface, which involves helixes A and C, and a subsequent conformational change in the protein that adopts a molten globule-like state to maximize the interaction between hydrophobic residues in these helixes and the lipid bilayer (21Halskau O. Froystein N.A. Muga A. Martinez A. J. Mol. Biol. 2002; 321: 99-110Crossref PubMed Scopus (70) Google Scholar). Svensson et al. (22Svensson M. Sabharwal H. Håkansson A. Mossberg A.K. Lipniunas P. Leffler H. Svanborg C. Linse S. J. Biol. Chem. 1999; 274: 6388-6396Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 23Svensson M. Håkansson A. Mossberg A.K. Linse S. Svanborg C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4221-4226Crossref PubMed Scopus (313) Google Scholar) have characterized an apoptosis-inducing conformer of human α-lactalbumin (human α-lactalbumin made lethal to tumor cells, i.e. HAMLET), that induces the death of tumor cells and immature cells, but does not harm healthy cells. As yet, the mechanism by which a larger protein as the HAMLET conformer of α-lactalbumin induces apoptosis is unknown, but both a partially unfolded conformation and a specific fatty acid as bound cofactor, oleic acid (18:1), are required for this new function of the protein. Conversion of α-lactalbumin to the apoptosis inducing form is achieved with both the protein derived from human milk whey and with recombinant protein expressed in Escherichia coli (23Svensson M. Håkansson A. Mossberg A.K. Linse S. Svanborg C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4221-4226Crossref PubMed Scopus (313) Google Scholar). It has also been recently found that the permeabilizing effect of HAMLET on the mitochondria with subsequent cytochrome c release, which may lead to activation of the caspase cascade and apoptotic death in transformed cells, is dependent on the oleic acid cofactor of HAMLET (24Kohler C. Gogvadze V. Håkansson A. Svanborg C. Orrenius S. Zhivotovsky B. Eur. J. Biochem. 2001; 268: 186-191Crossref PubMed Scopus (81) Google Scholar). The specificity of HAMLET for tumor cells leads the attention to the lipids involved in the recognition mechanism. It appears that the membrane composition is different in healthy cells and its corresponding tumor cells (25Hietanen E. Punnonen K. Punnonen R. Auvinen O. Carcinogenesis. 1986; 7: 1965-1969Crossref PubMed Scopus (51) Google Scholar, 26Chajes V. Lanson M. Fetissof F. Lhuillery C. Bougnoux P. Int. J. Cancer. 1995; 63: 169-175Crossref PubMed Scopus (44) Google Scholar, 27Chajes V. Hulten K. Van Kappel A.L. Winkvist A. Kaaks R. Hallmans G. Lenner P. Riboli E. Int. J. Cancer. 1999; 83: 585-590Crossref PubMed Scopus (120) Google Scholar, 28Monteggia E. Colombo I. Guerra A. Berra B. Cancer Detect. Prev. 2000; 24: 207-211PubMed Google Scholar). In human breast cancer tissue the amount of phospholipid has been measured to be 3.6-fold higher than in non-tumorous breast tissue (25Hietanen E. Punnonen K. Punnonen R. Auvinen O. Carcinogenesis. 1986; 7: 1965-1969Crossref PubMed Scopus (51) Google Scholar) and tumor cell membranes contain more anionic phospholipids and a different fatty acid composition (29Van Blitterswijk W.J. De Veer G. Krol J.H. Emmelot P. Biochim. Biophys. Acta. 1982; 688: 495-504Crossref PubMed Scopus (124) Google Scholar). Moreover, although the negatively charged phospholipids of the plasma membrane are usually segregated to the inner leaflet (30Cullis P.R. Hope M.J. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins and Membranes. 20. Elsevier Science Publishers B. V., Amsterdam1991: 1-41Google Scholar), the earliest sign of apoptosis is translocation of phosphatidylserine (PS) from the inner to the outer leaflet (31Vermes I. Haanen C. Steffens-Nakken H. Reutelingsperger C. J. Immunol. Methods. 1995; 184: 39-51Crossref PubMed Scopus (4607) Google Scholar). To further investigate the conformational changes accompanying the binding of α-lactalbumin to membranes, we have studied the interaction of BLA with liposomes of different composition. The conformation of the membrane-bound states of the protein was investigated by fluorescence spectroscopy, circular dichroism (CD), and 1H NMR. The interaction of BLA with the membrane seems to be mostly modulated by the nature, physical state, and charge of the major lipid components of the membranes, the glycerophospholipids (20Bañuelos S. Muga A. FEBS Lett. 1996; 386: 21-25Crossref PubMed Scopus (28) Google Scholar). In this study we have studied the interaction of BLA with liposomes made of different molecular forms of PS alone or equimolar mixtures of these lipids and egg yolk phosphatidylcholine (EYPC), thus with bilayers of different fluidity and charge density. Most of the previous studies on the interaction of BLA with model membranes have used liposomes containing negatively charged dioleoylphosphatidylglycerol (DOPG), although this phospholipid generally contributes to less than 1% to the total animal cellular phospholipids, except for the 2–5% found in lungs (30Cullis P.R. Hope M.J. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins and Membranes. 20. Elsevier Science Publishers B. V., Amsterdam1991: 1-41Google Scholar). Our results with the more relevant PS are compared with previous results obtained with mixtures of EYPC and DOPG. Materials—1,2-Distearoyl-sn-glycero-3-(phospho-l-serine) (DSPS), 1-stearoyl-2-oleoyl-sn-glycero-3-(phospho-l-serine) (SOPS), and 1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-l-serine) (POPS) were purchased from Avanti Polar Lipids (Alabaster, AL). EYPC, 1,2-dioleoylphosphatidylglycerol (DOPG), and bovine α-lactalbumin type III (holo-protein, calcium-saturated) were from Sigma. Deuterium oxide (99.9%) was from ICN Biomedicals Inc. (Costa Mesa, CA). Fura-2 was from Molecular Probes (Leiden, The Netherlands). Preparation of Unilamellar Lipid Vesicles—The lipids were dissolved in chloroform and mixed in a round-bottom glass flask to the desired proportions. The solvent was evaporated and the lipids freeze-dried overnight. The dried films were then dispersed in 20 mm citric acid/Na2HPO4, 0.1 m NaCl, pH 4.5, by gently mixing and small unilamellar vesicles (SUV), also referred to as liposomes in the text, were then prepared in a bath sonicator (Branson 1200, Bransonic, CT), operating at a nominal frequency of 20 kHz during 60–90 min at 4 °C. The temperature was maintained by continuous exchange of the chilled water. A highly homogeneous vesicle preparation with a diameter of 40 nm was obtained, as seen by electron microscopy and quasi-elastic light scattering using a Malvern Zetasizer (Malvern, United Kingdom). Electron microscopy revealed that SUV were unilamellar. Large unilamellar vesicles (LUV) (∼1 μm diameter) were prepared by extrusion as described (32Nerdal W. Gundersen S.A. Thorsen V. Hoiland H. Holmsen H. Biochim. Biophys. Acta. 2000; 1464: 165-175Crossref PubMed Scopus (65) Google Scholar). Binding of α-Lactalbumin to Liposomes by Ultracentrifugation—Protein solutions (7 μm) and liposomes were mixed in 1 ml of 20 mm citric acid/Na2HPO4, 0.1 m NaCl, pH 4.5–6.0, at the indicated lipid:protein ratios. Samples were allowed to equilibrate for 30 min at room temperature and were then centrifuged at 105,000 × g for 30 min at 4 °C. The protein concentration in the supernatant was determined spectrophotometrically, using the extinction coefficients of 28,500 m–1 cm–1 at 280 nm, pH 7.0 (33Ewbank J.J. Creighton T.E. Biochemistry. 1993; 32: 3694-3707Crossref PubMed Scopus (133) Google Scholar). To account for the sedimentation of the free protein, samples containing the same protein concentration in the absence of liposomes were treated and centrifuged under the same experimental conditions. Determination of Free Calcium Content—Calcium concentration was measured using the Ca2+ indicator fura-2 (34Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar) using a LS-50B PerkinElmer luminescence spectrometer, according to the product information manual from the manufacturer of fura-2 (Molecular Probes) and references therein. Calcium was measured in samples of BLA (0.1 mm) prepared in 20 mm citric acid/Na2HPO4, 0.1 m NaCl, pH 4.5 and 6.0, both in the presence and the absence of liposomes composed of EYPC: DOPG (1:1) and EYPC:SOPS (1:1) (7 mm phospholipid). Calcium was also measured after adjusting the pH of the samples to 6.0 in membrane-free protein fractions and in both membrane- and protein-free fractions prepared by ultrafiltration in Centricon 3 microconcentrators (Amicon) using 1 μm fura-2. Differential Scanning Calorimetry—Measurements were performed on a MicroCal VP-DSC differential scanning calorimeter (MicroCal, Inc.) with cell volumes of 0.5 ml at a scan rate of 60 °C/h. All buffer solutions were degassed under vacuum prior to use. Calorimetric cells were kept under an excess pressure of 207 kPa to prevent degassing during the scan. SUV (5 mm in phospholipid) prepared in a 20 mm citric acid/Na2HPO4, 0.1 m NaCl buffer, at the indicated pH, were used, and thermograms of buffer served as reference. When indicated, BLA was present at a concentration of 50–70 μm. Determination of the transition temperature (TC) and the half-widths of the transitions (TC½) was performed by curve fitting with the Origin TM software (MicroCal, Inc.). Fluorescence Spectroscopy—Fluorescence measurements were performed at 25 °C with a PerkinElmer luminescence spectrometer LS-50B with temperature regulation using quartz cuvettes with a light path of 5 mm. Samples contained 1 μm BLA in 20 mm citric acid/Na2HPO4, 0.1 m NaCl, pH 4.5 to 6.0, in the presence or absence of liposomes (300 μm in lipid). The fluorescence emission spectra of the protein were recorded in the 310–500 nm range with excitation at 295 nm using 3- and 5-nm band widths in the excitation and emission pathways, respectively. Protein-free blanks with and without liposomes of identical concentration and composition were subtracted. When indicated, the pH of the sample was adjusted from pH 4.5 to 6.0 by the addition of NaOH and incubation up to 30 min at 25 °C. Circular Dichroism (CD)—CD measurements were performed with a Jasco J-810 spectropolarimeter equipped with a PTC-348WI Peltier element for temperature control using quartz cells with path lengths of 1 mm. Samples contained 12.7 μm BLA in 20 mm citric acid/Na2HPO4, 0.1 m NaCl, pH 4.5 to 6.0, in the presence and absence of liposomes (882 μm in lipid) at the indicated temperature. Four consecutive wavelength scans between 195 and 260 nm were recorded for each CD spectrum and buffer blanks were subtracted. Protein thermal denaturation was monitored by following the changes in ellipticity at 222 nm, with a scan rate of 1 °C/min in the 10–95 °C temperature range. Mean residue ellipticity (Θ) was calculated from the formula Θ = ϵ/(10Cnl), where ϵ is the ellipticity (millidegrees), l is the path of the cuvette (cm), C is the protein concentration (mol/liter), and n is the number of amino acid residues in the protein (123 for BLA). Analysis of the data and determination of midpoint denaturation temperatures (Tm) of the protein were performed using the Standard Analysis program provided with the instrument. The amount of secondary structure elements was estimated with the CDNN program that applies a neural network procedure (35Bohm G. Muhr R. Jaenicke R. Protein Eng. 1992; 5: 191-195Crossref PubMed Scopus (1016) Google Scholar). Hydrogen-Deuterium Exchange and NMR Spectroscopy—Samples of BLA (1 mm final concentration, 550 μl) were prepared in 99.9% D2O-containing 20 mm citric acid/Na2HPO4, 0.1 m NaCl, pD 4.5, in the absence and presence of liposomes of different phospholipid composition to give a final lipid:protein molar ratio of 70. Total binding of the protein was controlled by ultracentrifugation and by the increase in fluorescence emission intensity (see above). Protons of BLA were then allowed to exchange with deuterium by incubation at 4 °C for 1 h, together with a reference sample that was prepared in the same buffer but in the absence of SUV. The pD was then adjusted to pD 6.0 by adding NaOH to release the protein from the membrane and NMR spectra were acquired. All NMR experiments were performed on a Bruker DRX 600 MHz spectrometer equipped with pulsed field gradient accessories. Band-selective homonuclear decoupled TOCSY (BASHD-TOCSY) experiments (36Krishnamurthy V.V. Magn. Reson. Chem. 1997; 35: 9-12Crossref Scopus (53) Google Scholar) were performed at a probe temperature of 308 K, to diminish adverse effects from high viscosity in the samples with liposomes. The spectra were acquired with a spectral width of 2.1 ppm in the evolution dimension (F1) and 14.985 ppm in the acquisition dimension (F2). The strength of the Gaussian cascade Q3 pulse was calibrated by setting its integral equal to a conventional hard π-pulse. The frequency offset of the soft pulse was adjusted to be [4.55-νwater]·600.13 Hz, where νwater is the water resonance frequency in ppm and 4.55 is the center of the excitation profile in the F1 dimension. The pulse program contained water suppression using pulse sculpting (37Hwang T.-L. Shaka A.J. J. Magn. Reson. Ser. A. 1995; 112: 275-279Crossref Scopus (1565) Google Scholar) and the "W5" modification of the 3-9-19 "Watergate" sequence (38Liu M. Mao X. Ye C. Huang H. Nicholson J.K. Lindon J.C. J. Magn. Res. 1998; 132: 125-129Crossref Scopus (470) Google Scholar). The number of scans was 96 and the time domain in F2 and F1 were 2048 and 32 complex points, respectively. Mixing time for the spin-lock field was set to 40 ms, the strength of the DIPSI-2 spin lock field was ≈6 kHz, and the recycling delay was set to 1.0 s. This set-up yielded an experimental time of 1 h and 8 min. Spectra processing was performed using the Xwinnmr (Bruker) software and volume of the cross-peaks in each of the BASHDTOCSY spectra was measured using Sparky 3.95 (39Goddard, T. D., and Kneller, D. G. (1999) SPARKY 3, University of California, San Francisco, CA www.cgl.ucsf.edu/home/sparky/)Google Scholar) by the sum over ellipsoid method. The resulting integrals of the αH-NH cross-peaks were divided by the volume of the non-exchanging cross-peak assigned to W26 H6–H7. Fluorescence and Differential Scanning Calorimetry Measurements—The pH-controlled reversible interaction of BLA with negatively charged liposomes of EYPC:DOPG (1:1) has been characterized by several spectroscopic techniques, including fluorescence spectroscopy (19Bañuelos S. Muga A. J. Biol. Chem. 1995; 270: 29910-29915Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). BLA free in solution shows an intrinsic fluorescence emission spectrum with λmax at about 330–331 nm both at pH 4.5 and 6.0 (Fig. 1) and on binding to liposomes of EYPC:DOPG at pH 4.5, the fluorescence intensity increases and the λmax red shifts to 339–340 nm (19Bañuelos S. Muga A. J. Biol. Chem. 1995; 270: 29910-29915Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). These changes have been interpreted as being the result of the transition of at least one of its four Trp residues to a more polar environment and the disappearance of tertiary interactions that quench the fluorescence in the native state (19Bañuelos S. Muga A. J. Biol. Chem. 1995; 270: 29910-29915Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). No spectral changes occur when liposomes (either LUVs or SUVs) of EYPC:DOPG are added to BLA at pH 6.0 and additional methods, such as ultracentrifugation, have corroborated that no binding takes place at this pH (19Bañuelos S. Muga A. J. Biol. Chem. 1995; 270: 29910-29915Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 21Halskau O. Froystein N.A. Muga A. Martinez A. J. Mol. Biol. 2002; 321: 99-110Crossref PubMed Scopus (70) Google Scholar). Incubation of the protein with SUVs at protein:lipid molar ratios ranging from 1:70 to 1:300 at pH 4.5 and 37 °C, and consequent ultracentrifugation of the samples, reveals that almost all BLA (>90%) associates to SUV made of EYPC:DOPG, EYPC:SOPS, EYPC: POPS, and EYPC:DSPS. Full binding of BLA was also measured after incubation with the same lipid bilayers at 25 °C, except for those composed of EYPC:DSPS, for which no binding was observed at this temperature. Liposomes (either LUVs or SUVs) of EYPC alone do not affect the emission fluorescence of BLA at either pH (Fig. 1). A red-shift and an increase in fluorescence intensity are also observed on binding of BLA to liposomes of EYPC:SOPS and EYPC:POPS, whereas a blue shift was observed for the interaction of the protein with liposomes of EYPC:DSPS at 37 °C (Fig. 1 and Table I).Table IIntrinsic fluorescence parameters for BLA bound to SUV of different lipid compositionsLipid compositionλmaxIF25 °C37 °C25 °C37 °CnmEYPC:DOPG339.0339.075.075.1EYPC:SOPS338.5338.553.052.3EYPC:POPS340.0339.534.934.5EYPC:DSPS336.0328.020.467.0No bilayer (BLA alone)331.0332.516.714.4 Open table in a new tab To check the thermotropic properties of these samples they were characterized by differential scanning calorimetry, and in Table II we have summarized the gel to liquid crystalline phase transition temperatures (TC) of the bilayers. It should be mentioned that we focused on the characterization of SUVs because the advantageous use of these liposomes over LUV in the spectroscopic experiments, where higher lipid concentrations are required, has been discussed elsewhere (21Halskau O. Froystein N.A. Muga A. Martinez A. J. Mol. Biol. 2002; 321: 99-110Crossref PubMed Scopus (70) Google Scholar). The values of TC obtained by differential scanning calorimetry for SUV containing pure PS species are consistent with data for the gel to liquid crystalline phase transition for LUV and MLV of the same phospholipids found in the LIPIDAT data base (40Caffrey M. Hogan J. Chem. Phys. Lipids. 1992; 61: 1-109Crossref PubMed Scopus (108) Google Scholar). The TC values for the mixtures of EYPC and different molecular species of PS in the absence of BLA are lower than for the pure PS species (Table II). The analysis of the pH dependence of TC, sensitive to the degree of ionization, the surface charge density, and the fluidity (41van Dijck P.W. de Kruijff B. Verkleij A.J. van Deenen L.L. de Gier J. Biochim. Biophys. Acta. 1978; 512: 84-96Crossref PubMed Scopus (276) Google Scholar, 42Phillips M.C. Ladbrooke B.D. Chapman D. Biochim. Biophys. Acta. 1970; 196: 35-44Crossref PubMed Scopus (227) Google Scholar), indicates that the PS species used in this study are negatively charged in the pH 4.5–6 range.Table IITemperature for the gel to liquid crystalline phase transition (TC) and half-width (TC½) of the transition obtained by differential scanning calorimetry for the SUV preparations studiedSampleNo BLABLA (60 μM)TCTC½TCTC½°C°CSOPS26.7 ± 0.76.8 ± 0.528.0 ± 0.66.9 ± 1.5EYPC:SOPS8.3 ± 0.515.8 ± 0.619.9 ± 0.816.7 ± 1.9POPS12.1 ± 0.11.5 ± 0.118.6 ± 0.10.9 ± 0.2EYPC:POPS4.1 ± 0.89.5 ± 0.715.1 ± 0.27.2 ± 0.5DSPSaNo apparent binding of the protein as measured by ultracentrifugation or other methods.67.6 ± 0.21.2 ± 0.269.0aNo apparent binding of the protein as measured by ultracentrifugation or other methods.1.7aNo apparent binding of the protein as measured by ultracentrifugation or other methods.EYPC:DSPS51.6 ± 0.112.9 ± 0.341.1 ± 0.210.5 ± 0.7a No apparent binding of the protein as measured by ultracentrifugation or other methods. Open table in a new tab When the pH was increased from 4.5 to 6.0, the fluorescence spectrum of BLA in the presence of liposomes essentially reverted to that for free BLA (Ref. 21Halskau O. Froystein N.A. Muga A. Martinez A. J. Mol. Biol. 2002; 321: 99-110Crossref PubMed Scopus (70) Google Scholar, Fig. 1, and data not shown), in accordance with the release of the protein from the bilayer. Measurement of the calcium content in solutions of BLA bound to liposomes of EYPC:DOPG and EYPC:SOPS at a lipid:protein molar ratio of 300:1 at pH 4.5 using the fluorescence properties of fura-2 (34Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar) showed that no calcium ion is released to the medium from the protein on binding to the bilayer, indicating that membrane-bound BLA most probably remains as holoenzyme. Moreover, BLA released from the membrane by the pH shift to pH 6.0 appears to be recovered largely (80%) as holoenzyme. The Association of BLA with Liposomes of Different Compositions Studied by CD—The far-ultraviolet spectrum of free BLA shows two minima at 208 and 222 nm, characteristic of proteins with large content of α-helical structure (Ref. 19Bañuelos S. Muga A. J. Biol. Chem. 1995; 270: 29910-29915Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar and Fig. 2A). The content of α-helix and β-extended structures in BLA estimated from the CD spectrum was 27 and 10%, respectively, in agreement with the content estimated from the crystal structure (43Pike A.C. Brew K. Acharya K.R. Structure. 1996; 4: 691-703Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). In the presence of liposomes of EYPC:DOPG, EYPC:SOPS, EYPC:POPS, and EYPC:DSPS at pH 4.5 and 10 °C, an increase was observed in the ellipticity of BLA (Fig. 2A), corresponding to larger apparent α-helix content than in free BLA (Table III). When the CD spectra were acquired at 37 °C, the negative ellipticity at 222 nm of BLA and the apparent α-helix c
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