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α-Synuclein Has a High Affinity for Packing Defects in a Bilayer Membrane

2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês

10.1074/jbc.m401076200

1083-351X

Brigitte Nuscher, Frits Kamp, Thomas Mehnert, Sabine Odoy, Christian Haass, Philipp J. Kahle, Klaus Beyer,

Parkinson's Disease Mechanisms and Treatments

A number of neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are characterized by the intracellular deposition of fibrillar aggregates that contain a high proportion of α-synuclein (αS). The interaction with the membrane-water interface strongly modulates folding and aggregation of the protein. The present study investigates the lipid binding and the coil-helix transition of αS, using titration calorimetry, differential scanning calorimetry, and circular dichroism spectroscopy. Titration of the protein with small unilamellar vesicles composed of zwitterionic phospholipids below the chain melting temperature of the lipids yielded exceptionally large exothermic heat values. The sigmoidal titration curves were evaluated in terms of a simple model that assumes saturable binding sites at the vesicle surface. The cumulative heat release and the ellipticity were linearly correlated as a result of simultaneous binding and helix folding. There was no heat release and folding of αS in the presence of large unilamellar vesicles, indicating that a small radius of curvature is necessary for the αS-membrane interaction. The heat release and the negative heat capacity of the protein-vesicle interaction could not be attributed to the coil-helix transition of the protein alone. We speculate that binding and helix folding of αS depends on the presence of defect structures in the membrane-water interface, which in turn results in lipid ordering in the highly curved vesicular membranes. This will be discussed with regard to a possible role of the protein for the stabilization of synaptic vesicle membranes. A number of neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are characterized by the intracellular deposition of fibrillar aggregates that contain a high proportion of α-synuclein (αS). The interaction with the membrane-water interface strongly modulates folding and aggregation of the protein. The present study investigates the lipid binding and the coil-helix transition of αS, using titration calorimetry, differential scanning calorimetry, and circular dichroism spectroscopy. Titration of the protein with small unilamellar vesicles composed of zwitterionic phospholipids below the chain melting temperature of the lipids yielded exceptionally large exothermic heat values. The sigmoidal titration curves were evaluated in terms of a simple model that assumes saturable binding sites at the vesicle surface. The cumulative heat release and the ellipticity were linearly correlated as a result of simultaneous binding and helix folding. There was no heat release and folding of αS in the presence of large unilamellar vesicles, indicating that a small radius of curvature is necessary for the αS-membrane interaction. The heat release and the negative heat capacity of the protein-vesicle interaction could not be attributed to the coil-helix transition of the protein alone. We speculate that binding and helix folding of αS depends on the presence of defect structures in the membrane-water interface, which in turn results in lipid ordering in the highly curved vesicular membranes. This will be discussed with regard to a possible role of the protein for the stabilization of synaptic vesicle membranes. α-Synuclein (αS) 1The abbreviations used are: αS, α-synuclein; ITC, isothermal titration calorimetry; CD, circular dichroism; DSC, differential scanning calorimetry; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; POPC, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleyl-sn-glycero-3-glycerol; BBSM, bovine brain sphingomyelin; SV, synaptic vesicles; βS, β-synuclein; wt, wild-type; apo, apolipoprotein. 1The abbreviations used are: αS, α-synuclein; ITC, isothermal titration calorimetry; CD, circular dichroism; DSC, differential scanning calorimetry; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; POPC, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleyl-sn-glycero-3-glycerol; BBSM, bovine brain sphingomyelin; SV, synaptic vesicles; βS, β-synuclein; wt, wild-type; apo, apolipoprotein. is a protein of 140 amino acids that has been identified as a major component of the intracytoplasmic fibrillar deposits (Lewy bodies) associated with idiopathic and inherited forms of Parkinson's disease (1Goedert M. Nat. Rev. Neurosci. 2001; 2: 492-501Crossref PubMed Scopus (1088) Google Scholar). The majority of cases are idiopathic, whereas mutations in the αS gene are known to be responsible for rare inherited, early onset variants of Parkinson's disease (2Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnson W.G. Lazzarini A.M. Duvoisin R.C. Di Iorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6600) Google Scholar, 3Krüger R. Kuhn W. Müller T. Woitalla D. Graeber M. Kösel S. Przuntek H. Epplen J.T. Schöls L. Riess O. Nat. Genet. 1998; 18: 106-108Crossref PubMed Scopus (3297) Google Scholar, 4Singleton A.B. Farrer M. Johnson J. Singleton A. Hague S. Kachergus J. Hulihan M. Peuralinna T. Dutra A. Nussbaum R. Lincoln S. Crawley A. Hanson M. Maraganore D. Adler C. Cookson M.R. Muenter M. Baptista M. Miller D. Blancato J. Hardy J. Gwinn-Hardy K. Science. 2003; 302: 841Crossref PubMed Scopus (3469) Google Scholar). Although the molecular mode of action of αS and of its homologs is as yet unknown, it was assumed that the protein modulates the dopamine neurotransmission by regulating synaptic vesicle (SV) mobilization from the presynaptic reserve pool (5Murphy D.D. Rueter S.M. Trojanowski J.Q. Lee V.M.-Y. J. Neurosci. 2000; 20: 3214-3220Crossref PubMed Google Scholar, 6Cabin D.E. Shimazu K. Murphy D. Cole N.B. Gottschalk W. McIlwain K.L. Orrison B. Chen A. Ellis C.E. Paylor R. Lu B. Nussbaum R.L. J. Neurosci. 2002; 22: 8797-8807Crossref PubMed Google Scholar) or directly regulating the dopamine metabolism (7Lee F.J.S. Liu F. Pristupa Z.B. Niznik H.B. FASEB J. 2001; 15: 916-926Crossref PubMed Scopus (381) Google Scholar, 8Perez R.G. Waymire J.C. Lin E. Liu J.J. Guo F. Zigmond M.J. J. Neurosci. 2002; 22: 3090-3099Crossref PubMed Google Scholar, 9Lotharius J. Barg S. Wiekop P. Lundberg C. Raymon H.K. Brundin P. J. Biol. Chem. 2002; 277: 38884-38894Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). However, attempts to identify a specific SV-binding protein, e.g. by protein cross-linking, have been unsuccessful so far. At a certain threshold concentration, αS tends to aggregate into amyloid fibrils (10Wood S.J. Wypych J. Steavenson S. Louis J.C. Citron M. Biere A.L. J. Biol. Chem. 1999; 274: 19509-19512Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar), whereas the homolog βS, which lacks a stretch of amino acids within the central portion of αS, has a much lower fibrillization propensity (11Serpell L.C. Berriman J. Jakes R. Goedert M. Crowther R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4897-4902Crossref PubMed Scopus (652) Google Scholar, 12Biere A.L. Wood S.J. Wypych J. Steavenson S. Jiang Y. Anafi D. Jacobsen F.W. Jarosinski M.A. Wu G.-M. Louis J.-C. Martin F. Narhi L.O. Citron M. J. Biol. Chem. 2000; 275: 34574-34579Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) and may even inhibit αS fibrillization (13Hashimoto M. Rockenstein E. Mante M. Mallory M. Masliah E. Neuron. 2001; 32: 213-223Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 14Uversky V.N. Li J. Souillac P. Millett I.S. Doniach S. Jakes R. Goedert M. Fink A.L. J. Biol. Chem. 2002; 277: 11970-11978Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 15Park J.Y. Lansbury Jr., P.T. Biochemistry. 2003; 42: 3696-3700Crossref PubMed Scopus (141) Google Scholar), indicating that the hydrophobic central part of αS is essential for its fibrillar aggregation (16Kahle P.J. Haass C. Kretzschmar H.A. Neumann M. J. Neurochem. 2002; 82: 449-457Crossref PubMed Scopus (77) Google Scholar). A recent study on the structure of mature fibrillar aggregates, using site-directed spin labeling, indicates that the N terminus of αS is less ordered than the central portion and that the C terminus is completely unfolded in the fibrillar aggregates (17Der-Sarkissian A. Jao C.C. Chen J. Langen R. J. Biol. Chem. 2003; 278: 37530-37535Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). According to circular dichroism (CD) and nuclear magnetic resonance spectroscopy, the monomeric αS protein is unfolded in buffer solution (18Weinreb P.H. Zhen W. Poon A.W. Conway K.A. Lansbury Jr., P.T. Biochemistry. 1996; 35: 13709-13715Crossref PubMed Scopus (1308) Google Scholar, 19Eliezer D. Kutluay E. Bussell Jr., R. Browne G. J. Mol. Biol. 2001; 307: 1061-1073Crossref PubMed Scopus (850) Google Scholar). In the presence of negatively charged phospholipid vesicles, however, the protein undergoes a transition into a partially α-helical state (20Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar). This has been attributed to the presence of positive amino acid charges in six imperfectly conserved repeats in the N-terminal region of the protein, which may account for the formation of a sided α-helix upon interaction with negatively charged membrane interfaces (19Eliezer D. Kutluay E. Bussell Jr., R. Browne G. J. Mol. Biol. 2001; 307: 1061-1073Crossref PubMed Scopus (850) Google Scholar, 20Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar, 21Jo E. McLaurin J. Yip C.M. St. George-Hyslop P. Fraser P.E. J. Biol. Chem. 2000; 275: 34328-34334Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar, 22Perrin R.J. Woods W.S. Clayton D.F. George J.M. J. Biol. Chem. 2000; 275: 34393-34398Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 23Chandra S. Chen X. Rizo J. Jahn R. Südhof T.C. J. Biol. Chem. 2003; 278: 15313-15318Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). In agreement with this notion, lipid binding and helix formation were barely detectable when αS was added to noncharged vesicles consisting of zwitterionic phospholipids in the liquid crystalline state (20Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar). It was also shown that the surface curvature of the vesicles is important for αS binding, i.e. there was little binding and helix folding in the presence of large unilamellar vesicles containing phosphatidylglycerol, in contrast to small unilamellar vesicles of the same composition (20Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar). This result may be not generally applicable, however, as αS binding to large unilamellar vesicles and even to multilamellar membranes containing negatively charged lipids other than phosphatidylglycerol was shown in later studies (24Narayanan V. Scarlata S. Biochemistry. 2001; 40: 9927-9934Crossref PubMed Scopus (158) Google Scholar, 25Jo E. Fuller N. Rand R.P. St George-Hyslop P. Fraser P.E. J. Mol. Biol. 2002; 315: 799-807Crossref PubMed Scopus (182) Google Scholar). Investigation of the aggregation kinetics revealed the formation of oligomeric intermediates of αS fibrillization (26Uversky V.N. Li J. Fink A.L. J. Biol. Chem. 2001; 276: 10737-10744Abstract Full Text Full Text PDF PubMed Scopus (929) Google Scholar). Such intermediates ("protofibrils") potentially incorporate into unilamellar vesicle membranes, resulting in vesicle permeabilization and metal ion influx (27Volles M.J. Lansbury Jr., P.T. Biochemistry. 2002; 41: 4595-4602Crossref PubMed Scopus (419) Google Scholar) or release of encapsulated dopamine (28Volles M.J. Lee S.J. Rochet J.C. Shtilerman M.D. Ding T.T. Kessler J.C. Lansbury Jr., P.T. Biochemistry. 2001; 40: 7812-7819Crossref PubMed Scopus (613) Google Scholar). Of particular interest is the observation that small amounts of an αS-dopamine adduct that forms under oxidizing conditions stabilize the protofibrillar intermediates (29Conway K.A. Rochet J.C. Bieganski R.M. Lansbury Jr., P.T. Science. 2001; 294: 1346-1349Crossref PubMed Scopus (982) Google Scholar). Thus, retardation of the protofibril-fibril transition may promote membrane permeation and release of cytotoxic amounts of dopamine from SV. Binding of αS to phospholipid membranes inhibited fibril formation (24Narayanan V. Scarlata S. Biochemistry. 2001; 40: 9927-9934Crossref PubMed Scopus (158) Google Scholar, 30Zhu M. Fink A.L. J. Biol. Chem. 2003; 278: 16873-16877Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), whereas fatty acid binding enhanced the formation of soluble αS oligomers (31Perrin R.J. Woods W.S. Clayton D.F. George J.M. J. Biol. Chem. 2001; 276: 41958-41962Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 32Cole N.B. Murphy D.D. Grider T. Rueter S. Brasaemle D. Nussbaum R.L. J. Biol. Chem. 2002; 277: 6344-6352Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 33Sharon R. Bar-Joseph I. Frosch M.P. Walsh D.M. Hamilton J.A. Selkoe D.J. Neuron. 2003; 37: 583-595Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 34Necula M. Chirita C.N. Kuret J. J. Biol. Chem. 2003; 278: 46674-46680Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Therefore, it will be important to pursue the investigation of the αS-lipid interaction with emphasis on the competition between helix folding and protofibrillar aggregation. The binding of αS to vesicular lipid membranes has been demonstrated by different techniques, including size exclusion chromatography, CD spectroscopy, and atomic force microscopy (20Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar, 21Jo E. McLaurin J. Yip C.M. St. George-Hyslop P. Fraser P.E. J. Biol. Chem. 2000; 275: 34328-34334Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar, 22Perrin R.J. Woods W.S. Clayton D.F. George J.M. J. Biol. Chem. 2000; 275: 34393-34398Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 28Volles M.J. Lee S.J. Rochet J.C. Shtilerman M.D. Ding T.T. Kessler J.C. Lansbury Jr., P.T. Biochemistry. 2001; 40: 7812-7819Crossref PubMed Scopus (613) Google Scholar). In the present study calorimetric techniques, i.e. isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC), provide another, more direct proof of protein-lipid interaction. The shape of the ITC curves depends characteristically on the binding mode, which can be either construed as a partition equilibrium or as specific binding with a limited number of saturable binding sites. Accordingly, a partition coefficient or a binding constant can be obtained, which yields the free energy and entropy of the binding reaction. Simultaneously with the calorimetric measurements, we have monitored the conformational transition of the protein to assess the contribution of helix folding to the overall heat release. The combination of ITC, DSC, and CD spectroscopy revealed an unexpected protein-lipid interaction, suggesting that not only negatively charged lipids but also defect structures in the membrane water interface are capable of inducing interfacial binding and helix folding of αS. These results will be discussed regarding the putative stabilization of intracellular, e.g. SV membranes by αS. Materials—Phospholipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleyl-sn-glycero-3-glycerol (POPG) were purchased from Avanti Polar Lipids (Alabaster, AL). Bovine brain sphingomyelin (BBSM) as well as other chemicals were from Sigma (Deisenhofen, Germany). Expression and Purification of Wild-type (wt) and A30P Mutant αS—The coding regions of human wt and A30P mutant αS (35Okochi M. Walter J. Koyama A. Nakajo S. Baba M. Iwatsubo T. Meijer L. Kahle P.J. Haass C. J. Biol. Chem. 2000; 275: 390-397Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar) were amplified by polymerase chain reaction with primers 5′-TTCATTACATATGGATGTATTCATGAAAGG-3′ and 5′-GGAATTCCATATGTTAGGCTTCAGGTTCGTAG-3′. Amplimers were subcloned into the NdeI site of pET-5a (Promega, Madison, WI), and constructs used to transform Escherichia coli BL21(DE3) pLys. All constructs were sequenced (Medigenomix, Munich/Martinsried, Germany). Bacterial cultures were induced with isopropyl-β-d-thiogalactopyranoside for 4 h, and lysed by freeze/thaw and sonication. After 15 min of boiling, the heat-stable 17,000 × g supernatant was loaded onto Q-Sepharose (Amersham Biosciences) and eluted with a 25–500 mm salt gradient. The pooled synuclein peak fractions were desalted by Superdex 75 gel filtration as described (36Kahle P.J. Neumann M. Ozmen L. Müller V. Odoy S. Jacobsen H. Iwatsubo T. Trojanowski J.Q. Takahashi H. Wakabayashi K. Bogdanovic N. Riederer P. Kretzschmar H.A. Haass C. Am. J. Pathol. 2001; 159: 2215-2225Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), and a final purification was achieved using a Sephacryl S-200 column (Amersham Biosciences). The protein concentration was determined photometrically at 275 nm using an extinction coefficient of ϵ = 5120 m–1 cm–1 (19Eliezer D. Kutluay E. Bussell Jr., R. Browne G. J. Mol. Biol. 2001; 307: 1061-1073Crossref PubMed Scopus (850) Google Scholar). Vesicle Preparation—Phospholipids, sphingomyelin, and lipid/cholesterol mixtures were dissolved in chloroform/methanol (1:1, v/v). The solvent was completely evaporated on a vacuum line, and the lipid films were resuspended in buffer (150 mm KCl, 20 mm phosphate, pH 7.2), followed by sonication with a tip sonicator (Branson Cell Disrupter B15). Typically, 1-ml volumes were sonicated above the respective phase transition temperatures (Tm) of the phospholipids for 20 min with a 30% duty cycle. The quality of the resulting small unilamellar vesicles was checked by dynamic light scattering. Sonicated vesicles prepared from DPPC or BBSM were stored at 45 °C and used within 2 h. The vesicles were rapidly cooled to the desired temperature below Tm prior to the titrations. Large unilamellar vesicles were prepared by extrusion of phospholipid suspensions using 100-nm polycarbonate membranes (LFM-100) and a LiposoFast™ extruder device (Armatis GmbH, Weinheim, Germany). ITC—A VP-ITC instrument equipped with a motor driven syringe (MicroCal, Amherst, MA) was employed for titration calorimetry. The volume of the calorimeter cell was 1.41 ml. Small volumes of the vesicle suspensions were injected with the computer-controlled syringe into the protein solution in the calorimeter cell. Typically, 40 injections of 7 μl each were executed and the integration of the calorimeter signals, base-line corrections, and normalization with respect to protein and lipid concentrations were done using MicroCal ORIGIN software. Only five to seven injections were necessary for the determination of the reaction heat by injection of protein into a suspension of lipid vesicles. Control titrations were performed, i.e. vesicles into buffer alone or protein into buffer alone, and the results were subtracted from the experimental data. The sigmoidal titration curves obtained with gel state vesicles were evaluated assuming independent saturable protein binding sites in the outer vesicle interface. First, the ligand concentration was equated with the total lipid concentration in the syringe, neglecting the aggregational state of the lipids. Second, N independent lipid binding sites on the protein were assumed. The fractional occupancy of lipid binding sites on the protein, Φ, and the free lipid and total protein concentrations [L] and [P]t, respectively, then yield the microscopic binding constant K′ = Φ/(1 – Φ)[L] and the total lipid concentration [L]t = [L] + NΦ[P]t. The fractional saturation of the protein is related to the total heat Q being released by Q = NΦ [P]t ΔH′ V0, where ΔH′ and V0 denote the molar enthalpy of ligand binding and the total volume of the cell. After elimination of [L] and Φ, the heat content of the cell can be expressed as a function of the total ligand concentration [L]t, where a = [L]t/N[P]t. Q=1/2N[P]tΔH′V0[1+a(1+K′−1)−{(1+a(1+K′−1))2−4a}](Eq. 1) Incremental heat values ΔQ = Qi – 1 – Qi were calculated and the experimental data was fitted by variation of N, K′, and ΔH′ according to the standard Marquard-Levenberg algorithm (37Bevington P.E. Data Reduction and Error Analysis for the Physical Sciences. McGraw-Hill, New York1969Google Scholar). Taking account of the spillover of liquid from the calorimeter cell during the titration (MicroCal tutorial guide), the "number of binding sites" N was identified with the total number of lipid molecules associated with one protein molecule, which yields the enthalpy (per mole of protein) ΔH0 = NΔH′, the association constant K = NK′, and the free energy (ΔG0 =–RT lnK) and entropy (ΔS0 = (ΔH0 –ΔG0)/T) of the binding reaction with respect to the protein. DSC—Differential scanning calorimetry was performed with a VPDSC instrument (MicroCal). Vesicles were used immediately after sonication. Base lines were corrected using the MicroCal software. CD—Spectra were recorded with a Jasco 810 spectropolarimeter (Jasco, Gross-Umstadt, Germany). A cuvette with 0.2-cm path length was filled with 500 μl of the protein solution. The proteins were titrated with phospholipid vesicles using a syringe for lipid addition and mixing of the protein and vesicle solutions. Control spectra obtained with vesicle suspensions in buffer alone were subtracted from the experimental spectra. Temperature control with an accuracy of ±0.5 °C in the cuvette was achieved with a heating/cooling accessory using a Peltier element. The helicity of the protein (i.e. the percentage of the entire protein sequence in an α-helical state) was obtained from the mean residue ellipticities, [Θ]222, according to Equations 2 and 3. Helicity (%)=100 fhelix(Eq. 2) fhelix=([Θ]222−[Θ]coil)/([Θ]coil−[Θ]helix)(Eq. 3) The mean residue ellipticities at 222 nm for the completely unfolded and for the completely folded protein were calculated from [Θ]coil = 640 – 45t and [Θ]helix = –40,000(1 – 2.5/n) + 100t (38Scholtz J.M. Qian H. York E.J. Stewart J.M. Baldwin R.L. Biopolymers. 1991; 31: 1463-1470Crossref PubMed Scopus (479) Google Scholar), where the parameters n and t denote the number of amino acids in the protein (140 for αS) and the temperature in degrees Celsius, respectively. Titration of αS with Negatively Charged Vesicles—It has been reported that phospholipid vesicles bearing negative lipid charges bind monomeric αS with a concomitant transition of the protein from a random coil into a partially helical conformation. For a quantitative assessment of the thermodynamics of this lipid-protein interaction, we employed ITC as shown in Fig. 1. Small unilamellar phospholipid vesicles obtained by sonication of an equimolar mixture of POPC and POPG were taken up into the syringe of the ITC instrument. Aliquots were titrated into the calorimeter cell containing a solution of wt-αS. The negative heat flow obtained after each vesicle injection (in μcal/s) indicates that the protein-lipid interaction is accompanied by an exothermic enthalpy. Integration of the individual calorimeter signals yielded the heat release per titration step (in μcal). In a parallel experiment, vesicles were injected into the buffer solution alone, which accounts for the effect of vesicle dilution. Enthalpy values in terms of kcal/mol of injected lipid were then obtained by dividing the integrated heat values by the respective molar amounts of lipid injected. As shown in Fig. 1B, the magnitude of the exothermic heat decreases monotonically with increasing phospholipid concentration in the calorimeter cell and vanishes when the total molar ratio exceeds 300 mol of phospholipid/mol of protein. The titration curve in Fig. 1 can be ascribed to a partition equilibrium, i.e. the amount of protein available for vesicle binding decreases with increasing vesicle concentration in the solution. The enthalpy for complete binding, ΔH0, was measured by titration of a dilute protein solution into a vesicle suspension in the calorimeter cell. Five injections were sufficient for an accurate determination. The data were further analyzed as described in detail by Seelig and colleagues (39Seelig J. Biochim. Biophys. Acta. 1997; 1331: 103-116Crossref PubMed Scopus (187) Google Scholar, 40Wieprecht T. Seelig A. Curr. Top. Membr. 2002; 52: 31-56Crossref Google Scholar). Briefly, the bound fractions, Xp, of the protein were calculated using the cumulative heat values, as shown in Equation 4, where δhi is the ith integrated calorimeter peak. Xp=∑i=1nδhi/ΔH0(Eq. 4) The unbound concentration of αS, cf, and the ratio of bound protein per total lipid in the calorimeter cell, Xb = n bP/n totL, was obtained from Xp (39Seelig J. Biochim. Biophys. Acta. 1997; 1331: 103-116Crossref PubMed Scopus (187) Google Scholar), which yielded the binding isotherm (Fig. 1B, inset). A simple partition equilibrium would result in a linear relation, Xb = Kp·cf, from which the binding constant Kp can be derived. A nonlinear relation was found, however, which must be attributed to electrostatic attraction of the protein by the negative charges at the vesicle surface, resulting in the effective protein concentration being higher close to the membrane-water interface than in bulk solution (Fig. 1, inset). For the binding of small peptides to charged vesicle membranes, the interfacial electrostatics were successfully taken into account using the Gouy-Chapman theory (41Seelig J. Nebel S. Ganz P. Bruns C. Biochemistry. 1993; 32: 9714-9721Crossref PubMed Scopus (99) Google Scholar). A quantitative analysis along these lines is not feasible for αS, as it requires knowledge of the effective protein charge and of the surface potential of the membrane. Qualitatively, it turns out that the binding ratio Xb increases in a nonlinear and nonsaturable manner, as can be expected for a partition equilibrium that is modulated by the surface potential of the vesicle membrane. The conformational rearrangement of αS as a consequence of the protein-vesicle interaction can be conveniently followed by CD spectroscopy (20Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar, 21Jo E. McLaurin J. Yip C.M. St. George-Hyslop P. Fraser P.E. J. Biol. Chem. 2000; 275: 34328-34334Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar, 22Perrin R.J. Woods W.S. Clayton D.F. George J.M. J. Biol. Chem. 2000; 275: 34393-34398Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 30Zhu M. Fink A.L. J. Biol. Chem. 2003; 278: 16873-16877Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 42Zhu M. Li J. Fink A.L. J. Biol. Chem. 2003; 278: 40186-40197Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar), i.e. a decreasing ellipticity at 222 nm is diagnostic for an increasing contribution of α-helical regions to the overall conformational equilibrium. Fig. 2 shows CD titrations of wt- and A30P-αS with vesicles consisting of binary mixtures of POPC and POPG at molar ratios 1:1 and 2:1, respectively. The CD data are presented in terms of the mean residue ellipticities ([Θ]222) to account for slightly different concentrations of the protein solutions in the cuvette. At low vesicle concentrations, the ellipticities decrease almost linearly as a function of the total lipid/protein molar ratio. The ratio of the initial slopes obtained with vesicles composed of POPC and POPG at molar ratios 2/1 and 1/1, respectively, is ∼1.4 for both the wt and the mutant protein. This compares favorably with the ratio of the POPG concentrations of the two different vesicle preparations (1.34), indicating that helix formation is a function of the interfacial charge density. More notably, the initial slope is significantly smaller for A30P-αS versus wt-αS for either vesicle composition, suggesting a lower affinity of the mutant protein for the charged membrane interface. In the presence of equimolar POPC/POPG vesicles, the ellipticities approach constant values at total lipid/protein molar ratios >200:1. Lipid/protein ratios resulting in maximum helix folding of the proteins were difficult to attain at the lower POPG content, as scattering from the more concentrated vesicle suspension resulted in unacceptably noisy CD spectra. Earlier work suggested that a highly curved membrane interface as obtained by sonication of phospholipid dispersions is required for vesicle binding and concomitant α-helix folding of monomeric αS (20Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar). Our calorimetric data are in line with this observation but are at variance with later reports (24Narayanan V. Scarlata S. Biochemistry. 2001; 40: 9927-9934Crossref PubMed Scopus (158) Google Scholar, 25Jo E. Fuller N. Rand R.P. St George-Hyslop P. Fraser P.E. J. Mol. Biol. 2002; 315: 799-807Crossref PubMed Scopus (182) Google Scholar). Neglig