Báo cáo khoa học: Effects of sphingomyelin, cholesterol and zinc ions on the binding, insertion and aggregation of the amyloid Ab1)40 peptide in solid-supported lipid bilayers ppt

Effects of sphingomyelin, cholesterol and zinc ions on the binding, insertion and aggregation of the amyloid Ab1)40 peptide in solid-supported lipid bilayers Savitha Devanathan1, Zdzislaw Salamon1, Goran Lindblom1, Gerhard Grobner2 and Gordon Tollin1 ă ă Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ, USA ˚ Department of Biophysical Chemistry, Umea University, Sweden Keywords Alzheimer’s disease; amyloid toxicity; microdomains; plasmon-waveguide resonance spectroscopy; rafts Correspondence G Tollin, Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721, USA Fax: +1 520 621 9288 Tel: +1 520 621 3447 E-mail: gtollin@u.arizona.edu (Received December 2005, revised 25 January 2006, accepted February 2006) doi:10.1111/j.1742-4658.2006.05162.x We utilized plasmon-waveguide resonance (PWR) spectroscopy to follow the effects of sphingomyelin, cholesterol and zinc ions on the binding and aggregation of the amyloid b peptide1)40 in lipid bilayers With a dioleoylphosphatidylcholine (DOPC) bilayer, peptide binding was observed, but no aggregation occurred over a period of 15 h In contrast, similar binding was found with a brain sphingomyelin (SM) bilayer, but in this case an exponential aggregation process was observed during the same time interval When the SM bilayer included 35% cholesterol, an increase of  2.5-fold occurred in the amount of peptide bound, with a similar increase in the extent of aggregation, the latter resulting in decreases in the bilayer packing density and displacement of lipid Peptide association with a bilayer formed from equimolar amounts of DOPC, SM and cholesterol was followed using a high-resolution PWR sensor that allowed microdomains to be observed Biphasic binding to both domains occurred, but predominantly to the SM-rich domain, initially to the surface and at higher peptide concentrations within the interior of the bilayer Again, aggregation was observed and occurred within both microdomains, resulting in lipid displacement We attribute the aggregation in the DOPC-enriched domain to be a consequence of lipid mixing within these microdomains, resulting in the presence of small amounts of SM and cholesterol in the DOPC microdomain When mm zinc was present, an increase of approximately threefold in the amount of peptide association was observed, as well as large changes in mass and bilayer structure as a consequence of peptide aggregation, occurring without loss of bilayer integrity A structural interpretation of peptide interaction with the bilayer is presented based on the results of simulation analysis of the PWR spectra The 39–42 amino acid residue amyloid b peptide (Ab) is a seminal etiologic factor in Alzheimer’s disease (AD), a member of the large family of neurodegenerative disorders with a common pathology in the form of aberrant protein folding [1–4] The unifying theme for all of these amyloidogenic diseases is the pathologi- cal conversion of specific proteins into toxic assemblies In the case of AD, its key substance, Ab peptide, is released as a soluble monomer, but seems to require a minimal level of aggregation to exert its neurotoxic action [3–9] In particular, soluble oligomeric and protofibrillar Ab structures are the primary toxic agents Abbreviations Ab, amyloid b1)40 peptide; AD, Alzheimer’s disease; AFM, atomic force microscopy; DOPC, dioleoylphosphatidylcholine; POPC, palmitoyloleoylphosphatidylcholine; PWR, plasmon-waveguide resonance; SM, brain sphingomyelin; TFA, trifluoroacetic acid; TFE, trifluoroethanol FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS 1389 Factors affecting Ab binding in lipid bilayers S Devanathan et al currently associated with the neuropathological events occurring in patients with AD [3,7–9] Nevertheless, globular and nonfibrillar Ab peptides are continuously released during normal metabolism in healthy people, with no problems observed, and therefore fundamental questions behind the toxic mechanism in AD are unsolved [10–12] Recently, the discovery of various soluble amyloid oligomers having a common structure, independent of their location, has brought new insight into possible mechanisms of toxicity [10] The inhibition of their toxicity by a common oligomer-specific antibody, connected to cell parts that are accessible by extra- and intracellular regions, has pointed strongly to cell membranes as a potential prime target [9,10,13– 15] This is not surprising because Ab has inherited a transmembrane domain from its precursor protein (a highly conserved integral membrane protein with a single transmembrane domain), providing it with an amphipathic nature, which makes it an ideal target for toxic events associated with neuronal membranes [5,13,16–22] The effects of Ab on membranes and lipid systems, and their possible roles in neurotoxicity, include changes in membrane fluidity, leading to membrane depolarization and disorder [23], membrane-mediated aggregation of Ab triggering neuronal apoptotic cell death [24], lipid peroxidation via H2O2 produced by Cu2+ reduction by Ab [22,25], and even the formation of calcium-permeable membrane ion channels [26] Owing to the amphipatic nature of Ab, a second process plays a potential key role in AD, namely the enhancing effects of specific neuronal membranes on Ab peptide conversion into toxic-acting oligomers Various lipid membranes have been shown to induce an electrostatically driven surface accumulation, followed by dramatically increased misfolding of Ab, at rates much higher than in a membrane-free environment [13–23,25–28] Membrane components, such as anionic lipids, gangliosides or cholesterol, were shown to be involved in various stages of Ab aggregation, and raft-like neuronal membranes seem to play a significant role in the regulation of Ab-production and its cytotoxic products [20–23,29,30] Interestingly, brain lipid composition in patients with AD is significantly altered, suggesting a link between lipid composition and increased susceptibility to neuronal cell death [16,31,32] Because of its amphipathic nature and the fact that patients with AD have altered neuronal lipid compositions [16,17], in the present study we investigated the role of raft-mimicking model neuronal membranes on the behavior of Ab peptide Using plasmon-waveguide resonance (PWR) spectroscopy [33,34] we elucidated features of the peptide–membrane inter1390 action, which might be important for raft membranedependent aggregation and neurotoxic action, in particular the presence of sphingomyelin, cholesterol and zinc ions Results and Discussion In order to characterize the interaction of Ab with lipid membranes, we used PWR spectroscopy to study the association with bilayers composed of single lipids, of binary lipid mixtures, or of a ternary mixture composed of dioleoylphosphatidylcholine (DOPC) ⁄ sphingomyelin (SM) ⁄ cholesterol (1 : : mole ratio), the latter in the presence and absence of added zinc ions This methodology has previously been used in our laboratory to characterize the composition of, the formation of, and insertion into, microdomains in bilayer membranes formed from binary mixtures of DOPC ⁄ SM and palmitoyloleoylphosphatidylcholine (POPC) ⁄ SM, both in : mole ratios [35,36] Interaction of Ab with single lipid bilayers Figure 1A,B shows that for a DOPC bilayer, interaction with increasing Ab concentrations produced small shifts to higher-incident angles in p-polarized and s-polarized spectra (11 and mdeg shifts, respectively, at a peptide concentration of lm in the aqueous cell compartment; s-polarized data not shown) The spectral shifts can be ascribed to an overall mass increase in the membrane as a result of peptide association with the bilayer These shifts followed a single hyperbolic curve with an apparent dissociation constant (KD) of 0.16 ± 0.02 lm (Fig 1B; Table 1) No further spectral changes were observed with time (over a time period of  15 h), indicating that no peptide aggregation occurred during this interval The addition of Ab to a lipid bilayer containing only SM again resulted in increasing spectral shifts to higher incident angles at peptide concentrations up to lm (Fig 2A,B) for both p- and s-polarized spectra (17 versus 14 mdeg, respectively; s-polarized data not shown) This is similar to what was observed for the DOPC bilayer, and occurred with a similar binding affinity (Fig 2B; Table 1) However, in the SM bilayer, after peptide binding and upon further equilibration with time (up to  15 h), a slow progressive increase in spectral position to higher-incident angles was observed These changes, which we attribute to peptide aggregation, are plotted in Fig 2C; they occurred exponentially with a half-time of  4.6 h (Table 2) We have obtained similar results to these with bilayers composed of DOPC and SM in a : mole ratio (data not shown) FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS S Devanathan et al Factors affecting Ab binding in lipid bilayers Table Amyloid b1)40 peptide (Ab) binding affinities (p-polarization) DOPC, dioleoylphosphatidylcholine; SM, sphingomyelin A 0.6 Reflectance Bilayer DOPC SM SM ⁄ cholesterol DOPC ⁄ SM ⁄ cholesterol DOPC-rich domain SM-rich domain 0.4 0.2 0.0 63.5 Resonance shifts (mdeg) B 63.6 63.7 Incident angle (deg) 63.8 KD (lM) 0.160 ± 0.02 0.220 ± 0.03 0.043 ± 0.01 0.370 ± 0.02 0.004 ± 0.001 (KD1) 0.110 ± 0.01 (KD2) DOPC ⁄ SM ⁄ cholesterol (+ mM Zn) DOPC-rich domain 0.450 ± 0.09 SM-rich domain 0.003 ± 0.001 (KD1) 0.022 ± 0.001 (KD2) a 15 10 0 Aβ (µM) Fig Binding of amyloid b1)40 peptide (Ab) to a dioleoylphosphatidylcholine (DOPC) bilayer (A) Plasmon-waveguide resonance (PWR) spectra for p-polarization are shown for a solid-supported DOPC bilayer before (curve 1) and after (curve 2; squares) the addition of Ab (5 lM bulk concentration in the aqueous cell compartment) The buffer used in the sample compartment was 10 mM Tris (pH 7.4) Experiments were performed at 25 ± 0.1 °C (B) Plot of p-polarized PWR resonance minimum spectral shifts as a function of the concentration of Ab added to the PWR sample compartment The binding data were fit by a single hyperbola (solid line), and the binding affinity values and the magnitude of the spectral shift are given in Table Interaction of Ab with binary lipid bilayers As shown by the data in Fig 3, and by the KD values in Table 1, Ab was bound fivefold more tightly to an SM bilayer containing cholesterol (1 : 0.35 mole ratio) and resulted in an approximately twofold larger magnitude in the spectral shifts obtained at lm peptide concentration (40 mdeg for p-polarization versus 27 mdeg for s-polarization; s-polarized data not shown) Again, time-dependent aggregation of the peptide occurred, with a half-time of  4.2 h (Fig 3C; Table 2) However, in this case the spectral changes involved shifts towards lower-incident angles (for Resonance shifts (mdeg)a 11 ± 17 ± 40 ± )6 ± 3±1 )15 ± )20 ± 20 ± )32 ± Extrapolated to infinite peptide concentration p-polarization, )23 mdeg and for s-polarization, )9 mdeg; s-polarized data not shown), contrary to that observed with the SM bilayer in the absence of cholesterol A spectral shift to smaller angles is caused by a decrease in refractive index As this parameter is proportional to mass density, we attribute this shift to a net loss in mass resulting from the removal of lipid molecules from the bilayer and transfer to the Gibbs border, which occurs upon peptide insertion into the bilayer and aggregation leading to lipid displacement (see below for further discussion) This contrasts with the process of peptide interaction and aggregation with the SM bilayer in the absence of cholesterol, where a net mass increase was observed as a result of peptide accumulation and possibly also lipid recruitment from the Gibbs border Interaction of Ab with ternary lipid bilayers For a ternary mixture composed of DOPC ⁄ SM ⁄ cholesterol (1 : : mole ratio), and using a PWR resonator design with a higher resolution that enabled the observation of membrane microdomains [35], two resonances were obtained corresponding to less-ordered thinner domains at lower-incident angles (DOPC enriched), and more ordered and more densely packed thicker microdomains (SM enriched) occurring at higher-incident angles (Fig 4A; cf ref 35 for evidence supporting this assertion) In this case, the Ab-binding process was biphasic (the initial phase is shown in the inset to Fig 4B), with an initial positive shift followed by a negative shift as the peptide concentration was increased, and occurred preferentially into the SM-rich microdomain The latter is shown by the larger shift observed for the resonance corresponding to this domain than for the DOPC-enriched domain (compare Fig 4B,C) FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS 1391 Factors affecting Ab binding in lipid bilayers S Devanathan et al B A 0.8 Resonance shifts (mdeg) 20 Reflectance 0.6 0.4 0.2 10 0.0 63.6 64.0 Incident angle (deg) 64.4 Resonance shifts (mdeg) C 45 30 15 10 Time (h) 15 Table Aggregation kinetics (p-polarization) DOPC, dioleoylphosphatidylcholine; SM, sphingomyelin Bilayer DOPC SM SM ⁄ cholesterol DOPC ⁄ SM ⁄ cholesterol DOPC-rich domain SM-rich domain DOPC ⁄ SM ⁄ cholesterol (+ mM Zn) a Half-times (h) Resonance shifts (mdeg)a – 4.6 ± 0.4 4.2 ± 0.3 4.5 3.7 t1 ¼ 0.53 t2 ¼ 2.96 t3 ¼ 17.9 ± ± ± ± ± 0.5 0.2 0.06 0.12 0.51 – 46 ± 65 ± )30 )58 )15 194 58 ± ± ± ± ± 5 Extrapolated to infinite time It is worth noting that accumulation of peptide at the microdomain surface, rather than insertion into its interior, is also possible We attribute the initial positive shift to a mass increase resulting from peptide binding to the bilayer surface, and the subsequent negative shift to peptide insertion into the bilayer and lipid displacement The higher spectral resolution allowed us to observe both of these processes in this experiment Consistent with the larger spectral shifts for the SM-rich 1392 Aβ (µM) Fig Binding and aggregation of Ab in a sphingomyelin (SM) bilayer (A) p-Polarized plasmon-waveguide resonance (PWR) spectra are shown for an SM bilayer before (curve 1) and after (curve 2; circles) the addition of Ab (5 lM bulk concentration in the aqueous cell compartment) Curve (triangles) shows the spectrum after equilibration for 15 h Other conditions were as in Fig (B) Plot of PWR spectral shifts induced by Ab binding to the bilayer with an increasing concentration of added peptide The data were fit by a single hyperbola, with the binding constant and total spectral shift given in Table (C) Plot of the time course of spectral changes for p-polarization associated with peptide aggregation A single exponential fit to the data is shown (solid line) with a half-time and total spectral shift as presented in Table microdomain, and thus a higher peptide concentration, the binding affinity of the peptide for this domain was approximately fourfold larger than for the DOPCenriched domain (Table 1) Note that a shift to lowerincident angles occurred for both microdomains, again indicating insertion of peptide within the bilayer, producing a less densely packed bilayer as a result of expulsion of lipid molecules That this occurred in both microdomains is probably a consequence of the fact that during microdomain formation a small amount of SM and cholesterol is mixed into the DOPC portion of the bilayer, and a small amount of DOPC and cholesterol is mixed into the SM-enriched microdomain [35] Peptide aggregation also occurred in this system, and within the first h the spectral changes occurred predominantly in the SM-enriched region (Fig 4A, curve 3), as evidenced by the smaller spectral shift that occurred in the DOPC-enriched domain Figure 4D shows the time course of resonance minimum shifts upon peptide aggregation At 15 h, the magnitude of the spectral changes was approximately twice as large for the SM-rich microdomain In the DOPC-rich microdomain, the half-time was 4.5 h, whereas it was slightly shorter in the SM-rich domain (Table 2) FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS S Devanathan et al Factors affecting Ab binding in lipid bilayers B Resonance shifts (mdeg) A Fig Binding and aggregation of Ab in a SM ⁄ cholesterol (1 : 0.35) bilayer (A) p-Polarized PWR spectra are shown for a bilayer before (curve 1) and after (curve 2; squares) Ab was added to the sample cell (5 lM bulk concentration in the aqueous compartment) Curve (triangles) shows the spectrum after equilibration for 15 h Other conditions were as in Fig (B) Plot of the spectral shifts associated with binding of the peptide to the bilayer as a function of added Ab concentration The binding constant was obtained by a hyperbolic fit to the data (solid line) with a KD value and total spectral shift as given in Table (C) Plot of the time course for p-polarization spectral changes associated with peptide aggregation The data were fit with a single exponential (solid line), and the half-time and total spectral shift are given in Table 0.6 0.4 Effect of Zn2+ on Ab interaction with a ternary lipid bilayer The binding of metal ions, such as Zn2+, to Ab peptides has been shown to facilitate peptide penetration of the hydrocarbon core of the membrane and subsequent aggregation [22,37] In order to test the effect of zinc addition in the present experiments on peptide binding and bilayer structural changes caused by aggregation, we used a DOPC ⁄ SM ⁄ cholesterol (1 : : mole ratio) bilayer in contact with a buffer containing mm Zn2+, present on both sides of the bilayer The spectral changes produced as a result of peptide binding and aggregation in the presence of zinc are shown in Fig 5A,B Control experiments showed that no PWR spectral changes occurred as a result of Zn2+ interaction with the lipid bilayer in the absence of peptide Figure 5A shows the spectral changes for p-polarization at various early time-points up to 200 after the addition of lm peptide The binding and aggregation process was observed to follow biphasic kinetics, with an initial shift to lower-incident angles that occurred in  30 min, followed at later time-points by a shift to higher angles accompanied by a decrease in 40 30 20 10 0 68.0 68.5 Incident angle (deg) Aβ (µM) C Resonance shifts (mdeg) Reflectance 0.8 40 20 -20 12 Time (h) 16 spectral amplitude for the lower-incident angle resonance (owing to the DOPC-rich domain), and an increase in amplitude and a large shift to higher angles for the resonance associated with the SM-rich domain (Fig 5A,B) These results suggest that a major change in bilayer structure occurred as a result of peptide insertion into the bilayer and aggregation The spectral changes are consistent with a high degree of mass accumulation occurring predominantly within the SM-rich microdomain However, it is important to note that the bilayer remained intact (i.e no large holes were formed that exposed resonator surface to the aqueous phase) This is evidenced by the fact that no resonances were observed corresponding to the bare prism surface in direct contact with the aqueous medium The binding isotherms for peptide association with the bilayer are shown in Fig 5C,D, with the binding constants given in Table Again, the initial binding process occurred with high affinity (inset to Fig 5C), similar to that observed in the absence of zinc However, in this case the second phase of binding had a fivefold higher affinity than in the absence of zinc (Fig 5C; Table 1) The peptide interacted with the FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS 1393 Factors affecting Ab binding in lipid bilayers S Devanathan et al Fig Binding and aggregation of Ab in a DOPC ⁄ SM ⁄ cholesterol (1 : : 1) bilayer Spectra were obtained using a high-resolution sensor Other experimental conditions are as given in the legend to Fig (A) p-Polarized PWR spectra are shown for the membrane before (curve 1) and after (curve 2; squares) addition of the peptide (5 lM bulk concentration in the aqueous cell compartment) Based on previous results [35], the resonance at smaller-incident angles is ascribed to a DOPC-enriched microdomain, and the resonance at larger incident angles to an SM-enriched microdomain The initial addition of peptide resulted in binding to both microdomains, but to a larger extent in the SM-rich domain Spectra are also shown after the sample had equilibrated for h (curve 3; circles) and 15 h (curve 4; triangles) These changes are ascribed to peptide aggregation (B,C) Plots of initial peptide binding, resulting in shifts to larger angles (inset to B) and binding at higher concentrations resulting in shifts to smaller angles Binding to the SM-rich domain is shown in (B) and to the DOPC-rich domain in (C) Data were fit with three hyperbolic curves (solid lines), yielding the affinity constants and spectral shifts in Table (D) Plots of the time course of spectral changes for the DOPC-rich microdomain (open triangles) and the SM-rich microdomain (closed triangles) associated with peptide aggregation Solid lines correspond to single exponential fits, with half-times and total shifts given in Table DOPC-rich domain with a single KD value 20-fold weaker than with the SM-rich domain, and with a smaller spectral shift (Fig 5D) The kinetics of the spectral changes are shown in Fig 6, and the half-times obtained by curve fitting are given in Table These reveal at least three kinetic phases: two at early time-points with half-time values of 0.53 ± 0.06 h and 2.96 ± 0.12 h; and a third, slower, phase corresponding to a half-time of 17.9 ± 0.5 h Thus, the mechanism by which aggregation occurs is quite complex in this system, and further studies will be necessary to obtain mechanistic insights 1394 These results are consistent with the reported effect of zinc on the formation of a helical peptide structure that facilitates insertion and pore formation as a result of aggregation [22,37,38] Furthermore, the atomic force microscopy (AFM) studies of Lin et al [39], on solidsupported bilayers, have shown that the interaction of Ab results in the formation of conducting ion channels that have rectangular or hexagonal shapes with four or ˚ six subunits, are 80–120 A in diameter and protrude ˚ from the bilayer surface The large PWR spec 10 A tral changes shown in Fig are consistent with structures of this magnitude inserted into the bilayer FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS S Devanathan et al Factors affecting Ab binding in lipid bilayers Fig Binding and aggregation of Ab in a DOPC ⁄ SM ⁄ cholesterol (1 : : 1) bilayer in the presence of mM zinc ions Spectra were obtained using a high-resolution sensor Other experimental conditions were as in Fig (A) and (B) p-Polarized PWR spectra obtained for the bilayer in the presence of mM Zn ions, as a function of time, after the addition of Ab (5 lM bulk concentration in the sample cell) Initial binding occurred mainly in the SM-rich microdomain (data not shown) With time, additional resonance shifts occurred, mainly in the SM-rich microdomain, along with large changes in amplitude The resonance for the DOPC-rich domain diminished in intensity, whereas that for the SMrich domain increased in intensity and large shifts to longer angles occurred Note that the t ¼ 208 resonance is repeated in both panels for reference purposes (C,D) Plots of initial peptide binding, resulting in shifts to larger angles (inset to C) and binding at higher concentrations resulting in shifts to smaller angles Binding to the SM-rich domain is shown in (C) and to the DOPC-rich domain in (D) Data were fit with three hyperbolic curves (solid lines) to yield affinity constants and spectral shifts as given in Table Initial insertion into the SM-rich domain was followed by incorporation into both microdomains Spectral simulation and structural modeling The purpose of PWR spectral simulation is to quantify the changes in the optical properties of the membrane that occur during the processes of lipid bilayer–peptide binding and subsequent time-dependent peptide aggregation This can provide insights into the changes in mass density and structure of the system caused by these events The simulation procedure is described in detail in our previous publication [35] Briefly, it is based on Maxwell’s equations that provide an analytical relationship between experimental spectral parameters and the optical properties of the bilayer, the latter defined by refractive index, n, extinction coefficient, k, and thickness, t These three parameters can provide a unique fit to the experimental spectra (In these experiments, the k-value is caused by light-scattering effects and will be ignored.) In the present analysis, based on the structure of the peptide and the literature data, we have assumed a working model that allows the peptide to either partially penetrate the lipid membrane with its hydrophobic tail (i.e residues 29–40), or to stay on the surface of the bilayer membrane without significant penetration This generates two separate layers, composed of peptide and of the lipid bilayer Using this two-layer model we have been able to quantify how much of the peptide molecule protrudes beyond the bilayer–water interface, and how much the lipid FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS 1395 Factors affecting Ab binding in lipid bilayers S Devanathan et al Reflectance 0.95 0.85 0.75 61.54 Fig Plot of the time course of spectral changes occurring in the SM-rich domain The solid line is a fit to the data with three exponentials; half-times and spectral shifts are given in Table membrane properties have been changed by the peptide–bilayer interaction This is accomplished by evaluation of the optical parameters for the lipid layer before and after binding of peptide and subsequent aggregation, and of the layer composed of peptide (or a portion of the peptide) that extends beyond the lipid surface Both p- and s-polarized data were used in such simulations Figure shows an example of the simulated spectra superimposed on the experimental spectra, obtained with s-polarized red light (k ¼ 632.8 nm) The optical parameters resulting from such simulations for bilayers composed of DOPC, of SM and of SM ⁄ cholesterol are summarized in Tables 3–5 for the binding and aggregation processes The error limits shown in these tables correspond to the standard errors obtained from the fitting procedure It must be noted that we have not been able to satisfactorily simulate the spectra obtained with the DOPC ⁄ SM ⁄ cholesterol bilayers, with and without zinc, using the high-resolution resonator This requires additional information about the effects of varying amounts of cholesterol on DOPC and SM bilayers, which we not have at present Thus, further experiments are needed in order to accomplish this Therefore, for the time being we have limited our discussion to qualitative aspects of Ab binding and aggregation in this system, as presented above There are several important conclusions that can be obtained from the values in Tables 3–5 First, the bilayer membranes consisting of DOPC, SM, or SM ⁄ cholesterol have quite different optical parameters 1396 61.64 61.74 Incident angle, deg Fig Examples of simulated spectra (solid lines) superimposed on experimental spectra (symbols), obtained with a SM ⁄ cholesterol membrane using s-polarized red light Spectra are shown for the membrane before (curve 1) and after (curve 2) addition of peptide Curve was obtained after aggregation Table Optical parameters of lipid bilayers prior to Ab binding DOPC, dioleoylphosphatidylcholine; np, p-polarized refractive index; ns, s-polarized refractive index; SM, sphingomyelin; tav, average thickness Bilayer np (± 0.003) ns (± 0.002) tav (nm) (± 0.1) DOPC SM SM ⁄ cholesterol 1.460 1.520 1.555 1.445 1.450 1.522 5.0 5.8 6.2 before peptide has been bound (Table 3) This has been observed previously for DOPC and SM bilayers [35] and discussed in the context of the membrane structure and properties The present work has now extended this to an SM bilayer containing 0.35 mole percent cholesterol Thus, the refractive indices clearly indicate that the bilayer consisting of DOPC is much less densely packed with lipid molecules than that of either SM or SM ⁄ cholesterol (note that refractive index is proportional to mass density, i.e molecules per unit surface area) The lipid-packing density influences the ordering of hydrocarbon tails, resulting in a higher degree of order in the latter two membranes, and this difference is reflected in an increased thickness of both membranes In addition, it is clear that the presence of cholesterol significantly increases both the packing density of lipid molecules (as shown by the increase in refractive index) as well as the thickness FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS S Devanathan et al Factors affecting Ab binding in lipid bilayers Table Optical parameters of lipid and peptide layers after amyloid b1)40 peptide (Ab) binding chol, cholesterol; DOPC, dioleoylphosphatidylcholine; np, p-polarized refractive index; ns, s-polarized refractive index; SM, sphingomyelin; tav, average thickness Lipid layer (after binding) Peptide layer (after binding) Bilayer np (± 0.003) ns (± 0.002) tav (nm) (± 0.2) np (± 0.005) ns (± 0.005) tav (nm) (± 0.2) DOPC SM SM ⁄ chol 1.460 1.520 1.555 1.445 1.450 1.522 5.0 5.8 6.2 1.340 1.365 1.420 1.340 1.365 1.400 3.0 3.0 4.7 Table Optical parameters of lipid and peptide layers after amyloid b1)40 peptide (Ab) aggregation chol, cholesterol; DOPC, dioleoylphosphatidylcholine; np, p-polarized refractive index; ns, s-polarized refractive index; SM, sphingomyelin; tav, average thickness Lipid layer (after aggregation) Peptide layer (after aggregation) Bilayer np (± 0.003) ns (± 0.002) tav (nm) (± 0.2) np (± 0.005) ns (± 0.005) tav (nm) (± 0.2) DOPC SM SM ⁄ chol No aggregation 1.535 1.470 No aggregation 1.465 1.460 No aggregation 5.8 6.2 No aggregation 1.365 1.420 No aggregation 1.350 1.400 No aggregation 3.0 4.7 of the SM bilayer membrane Therefore, peptide binding is occurring to three different membrane structures in these studies The data presented in Table show that the binding interaction of the peptide with the three membranes creates different effects within the lipid layer and in the peptide layer Although the peptide layers in both the DOPC and SM membranes are similar in thickness, indicating that the peptide protrusions from the bilayer surface are similar, they differ in the values of the refractive indices, indicating a higher peptide surface mass density in the case of the SM membrane as compared with the DOPC membrane It is also important to note that, in both of these cases, the values of the refractive indices are the same for both polarizations This strongly supports the idea that the internal structures of the peptide layers are quite isotropic, without any significant degree of long-range molecular order Based on this, one can conclude that these layers are formed from anisotropic molecular structures which are arranged in the peptide layer in a random way, thereby creating an average isotropic distribution of conformations It is worth noting that such anisotropies can result from either molecular conformation or molecular orientation, or both; these cannot be distinguished by the present methods In none of the three membrane systems did the initial binding of peptide cause any significant changes in the lipid bilayer parameters (Table 4) This suggests that the peptide was not anchored deeply within these membranes, indicating that the bulk of the peptide mass remained largely on the surface of the membrane However, the peptide layer was appreciably different for the SM ⁄ cholesterol membrane, having a higher mass density, a significant anisotropy (np > ns), and a larger thickness Thus, more peptide was bound when cholesterol was included in the bilayer, and the structure of the peptide layer was different In order to explain the anisotropic nature of the peptide layer, it seems reasonable to assume that some of bound peptides had an extended conformation (b-sheet like) and bound with their long axis perpendicular to the plane of the lipid membrane Furthermore, the 4.7 nm thickness of the peptide layer indicates that the hydrophobic tail of the peptide must be buried within the lipid bilayer This type of arrangement of the bound peptide is in agreement with previous studies showing that the addition of cholesterol to a DOPC ⁄ SM bilayer converted a b-sheet Ab form into a-helix structures, a change that was necessary to allow incorporation of the C-terminal tail into the membrane [40] Such incorporation is probably driven by hydrophobic interactions caused by the high nonpolar amino acid content in the C terminus of Ab A hydrophobic peptide segment would be expected to have an orientation and anisotropic refractive index values similar to both the fatty acyl tails of the lipid and the cholesterol molecule, and thus would not produce much change in the lipid portion of the bilayer However, the peptide layer would be appreciably altered by such a conformational change of the peptide It is generally accepted that carefully prepared fibrilfree Ab in an aqueous environment exists mainly as monomers, dimers, trimers, and tetramers, in a rapid equilibrium, within which the predominant secondary structural element is random coil with smaller amounts FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS 1397 Factors affecting Ab binding in lipid bilayers S Devanathan et al of b-sheet, and even smaller amounts of a-helix [41] We therefore assume that the peptide layer on the DOPC and SM membrane surface consists of such a mixture of oligomers and secondary conformations This accounts for the isotropic nature of this layer in these two systems It is interesting, however, to note that both the binding affinity and the amount of accumulated peptide (i.e mass density) were significantly higher with the SM bilayer than with the DOPC bilayer This is presumably a result of differences in both the type of lipid molecule (i.e sphingolipid versus glycerolipid), as well as the topology and morphology of the surfaces of each of the bilayers This requires further study The differences between the membranes increased further with time after the initial binding process As noted above, and as shown in Table 5, after 15 h there were no significant changes in either the bilayer or the peptide layer with the DOPC membrane (i.e no peptide aggregation occurred) In contrast, changes occurred in both of these layers with the SM membrane Thus, the surface mass density of the peptide layer decreased, as reflected by the smaller value of ns, and a significant refractive index anisotropy was induced (np > ns) A corresponding increase in the mass density in the lipid bilayer occurred, without a significant change in anisotropy These results clearly indicate that some peptide mass was inserted into the lipid membrane, and that the mass distribution within the peptide layer was altered In order for the latter to occur, a significant conformational rearrangement must take place, involving either creating more anisotropic conformations or reorganizing the existing anisotropic conformations so as to create long-range molecular order, or both A plausible interpretation of these effects is that some of the surface-bound peptide (possibly monomeric forms with a b-sheet structure) created higher oligomeric structures that inserted their hydrophobic tails into the interior of the membrane We suggest that this process was favored, in the case of the SM membrane, because the surface mass density (i.e concentration) of peptide was much higher than with the DOPC membrane As can be seen from the data presented in Table 5, the aggregation of the peptide in the lipid membrane consisting of SM ⁄ cholesterol resulted in large decreases in the refractive index parameters for the lipid layer This indicates a significant expulsion of lipid mass (presumably SM molecules) from the bilayer Thus, the aggregates formed in the presence of cholesterol must have a structure that occupies a larger fraction of the bilayer volume than the unaggregated peptide, in contrast to those formed in the absence of cholesterol 1398 This suggests that cholesterol may have been incorporated into these aggregated peptide structures, which requires further study It is also worth noting that the insertion and aggregation of Ab with membranes containing at least 30% cholesterol has been shown to form channel-like structures [42] This is consistent with our observation of lipid removal from the bilayer upon peptide insertion and aggregation in the SM ⁄ cholesterol system In summary, the data presented here provide strong support for a mechanism in which peptide aggregation requires an SM-rich environment and occurs largely on the bilayer surface in the absence of cholesterol The presence of cholesterol facilitates peptide insertion into the bilayer and promotes the aggregation process This leads to the formation of a less densely packed bilayer The presence of Zn2+ also enhances insertion and aggregation, and promotes large bilayer structural changes by peptide aggregates resulting in a more porous membrane The large effects of SM and cholesterol on peptide insertion and aggregation are consistent with the reported occurrence of the amyloid precursor protein and Ab in neuronal membrane rafts [20– 23,29,30] Experimental procedures Materials Solid-supported lipid bilayers (DOPC, SM, cholesterol and their mixtures; Avanti Polar Lipids, Birmingham, AL, USA) were made using solutions of either the single lipid components or mixtures containing various molar ratios of lipids (10 mgỈmL)1 total lipid concentration) in butanol ⁄ squalene (10 : 0.1, v ⁄ v) The buffer solution in contact with the bilayer in the sample cell for all the experiments was 10 mm Tris (pH 7.4) at 25 °C, either in the absence of additional salt ions or in the presence of mm Zn2+ ions The 40-residue amyloid peptide (Ab1)40) was synthesized by standard solid-phase Fmoc chemistry, subsequently purified by HPLC and found to be over 90% pure, and characterized by MALDI-TOF mass spectroscopy It was stored in a lyophilized form by a combined trifluoroacetic acid (TFA) ⁄ trifluoroethanol (TFE) protocol to keep it in a monomeric form prior to further use [19,28] PWR spectroscopy The principles of PWR spectroscopy have been thoroughly described in previous publications [34–36,43,44] Here, we will briefly review those aspects that are especially relevant to the present work Figure shows a schematic diagram of a PWR spectrometer Resonances can be excited with FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS S Devanathan et al Factors affecting Ab binding in lipid bilayers Formation of lipid membranes and peptide incorporation Fig Schematic diagram of a PWR apparatus A lipid bilayer with inserted peptides is shown immobilized on the resonator surface The presence of a silica layer allows excitation by both p-polarized and s-polarized light Excitation produces an evanescent electromagnetic field localized on the outer silica surface; molecules immobilized on this surface interact with the field causing spectral changes Adapted from a previous publication [44] light whose electric vector is polarized either perpendicular (p) or parallel (s) to the resonator surface, and can be used to probe the properties (refractive index, n, extinction coefficient at the excitation wavelength, k, and thickness, t) of molecules immobilized on the silica layer that coats a thin silver film deposited on the surface of a glass prism [33] This allows the characterization of the optical properties of uniaxially ordered anisotropic systems, such as proteolipid membranes, that are deposited on this surface, involving changes in surface mass density (i.e the amount of mass per unit surface area), in the spatial mass distribution (i.e the internal organization of the membrane, including molecular anisotropy and the long range molecular order of the bilayer), and in protein ⁄ lipid conformation occurring as a consequence of molecular interactions The PWR spectrum can be described by the depth, the half-width and the angular position of the resonances, which are determined by the optical characteristics of the sensor and the immobilized molecules at the plasmon excitation wavelength Molecular interactions or composition changes occurring at the surface are detected as changes in these spectral characteristics in real time Thus, PWR provides a means to directly measure the binding, insertion and aggregation of molecules, either at the lipid bilayer surface or upon incorporation into the bilayer The principles of creating a self-assembled single solid-supported planar lipid bilayer membrane on a PWR resonator surface have been described in previous publications [45,46] Here, we will briefly review those principles Bilayers were prepared by spreading a small amount of the appropriate lipid solution in the butanol ⁄ squalene solvent onto a mm orifice in a Teflon block, separating the silica surface of the PWR resonator from the aqueous compartment The hydrated silica surface attracts the polar head groups of the lipid to form a monolayer with the hydrocarbon tails oriented towards the excess lipid solution Bilayer formation is spontaneously initiated when the sample compartment is filled with an aqueous buffer solution, resulting in a thinning process to form the second monolayer A plateau-Gibbs border, consisting of an annulus of excess lipid solution, anchors the bilayer to the Teflon block This border allows lipid molecules to be transferred into or out of the orifice to accommodate structure ⁄ conformation changes that occur upon peptide insertion into the bilayer and subsequent aggregation The resonator surfaces used in these studies were of two designs – low resolution and high resolution – which varied in the intrinsic linewidth of the PWR resonances In order to observe membrane microdomains with appropriate lipid mixture compositions (containing equimolar amounts of DOPC, SM and cholesterol), a resonator that was specifically designed to obtain higher-resolution spectra was used [35] With this PWR system, the three-component bilayer spectra displayed two overlapping resonances These were shown previously, by spectral simulation [35], to be the result of lateral segregation of lipid molecules that spontaneously form membrane microdomains, with the more ordered thicker domains (SM-rich) producing resonances at higher-incident angles compared with the more disordered thinner domains (DOPC-rich), which produced resonances at lower-incident angles [35] The microdomains were estimated to be larger than 100 nm in diameter (note that the laser beam is  0.1 mm in diameter and the bilayer is  mm in diameter) It is also important to note that these microdomains consisted of lipid mixtures, with the majority component as indicated Bilayers composed of a single lipid generated a single resonance that could be observed using the standard low-resolution PWR resonator system When the standard resonator was used with the mixed bilayers, again a single resonance was observed, as a consequence of the lower spectral resolution of the system Some variability is expected in the self-assembling process and in the bilayer distribution of such microdomains from one experiment to another (i.e in the ratio between the lipid ordered and disordered phases), as well as in the sampling of such microdomains by the laser beam [35] This can result in variability in the spectral line shape from FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS 1399 Factors affecting Ab binding in lipid bilayers S Devanathan et al experiment to experiment (compare, for example, Fig 4A with Fig 5A) The diffusion of the microdomains within the bilayer is relatively slow and occurs in the order of minutes [47,48] PWR spectra were taken at regular intervals to follow the equilibration processes involved in bilayer formation occurring on the resonator surface After bilayer membrane equilibration, lyophilized TFA ⁄ TFE-pretreated Ab, freshly dissolved in aqueous buffer, was added in small (microliter) aliquots to the aqueous compartment of the PWR cell (total volume  mL), and binding of peptide to the bilayer was observed as a change in the resonance position minimum, with time, for both p- and s-polarization In all experiments, the total Ab concentration in the sample cell after these additions was lm The binding process reached equilibrium  after peptide addition Inasmuch as time-dependent aggregation of Ab is known to occur in aqueous media [41], it is not clear what the oligomeric state of the peptide was prior to being bound to the bilayer However, in a control experiment, the peptide was initially dissolved in dimethylsulfoxide and freshly prepared aliquots of this solution were added to the PWR cell, without producing much change in the subsequent behavior from that observed with the aqueous peptide solutions As previous work has shown [49], TFA ⁄ TFE-pretreated Ab is monomeric in dimethylsulfoxide Apparent peptide KD values were obtained from hyperbolic fits to plots of the resonance shift versus added peptide concentration; values reported in Table correspond to averages of the data for p- and s-polarization, which are expected to give the same values For membranes obtained with lipid mixtures containing DOPC ⁄ SM ⁄ cholesterol, Ab at lower concentrations preferentially inserted into the SM-rich microdomains, although at higher concentrations the peptide incorporated into both microdomains After the peptide concentration in the sample compartment reached lm, the experiment was allowed to proceed overnight (‡ 15 h) and monitored to follow aggregation of the peptide; PWR spectra were taken at regular intervals Note that the aggregation process occurred much more slowly (hours) than the initial peptide binding (minutes) The observed time course of aggregation was consistent with previous observations using other technologies [17,20,29] Note that PWR spectral changes not directly monitor peptide aggregate formation; rather, aggregation is detected by changes in the anisotropic ordering of peptide molecules and influences on the lipid bilayer structure This has the advantage of allowing information to be obtained concerning such structural modifications In order to determine whether aggregation required additional peptide to be recruited from the aqueous medium, some experiments were carried out in which the excess peptide in solution was removed by washing with fresh buffer subsequent to peptide binding to the bilayer These experiments resulted in spectral shifts (owing to aggregation) with time that were approximately the same magnitude as those obtained with- 1400 out washing Thus, we conclude that aggregation proceeded using peptide that was already bound to the bilayer All PWR experiments reported here were carried out using a Beta-PWR instrument (Proterion Corp., Piscataway, NJ, USA) using either 543.5 nm or 632.8 nm wavelength excitation Both wavelengths yielded similar results As performed in previous studies [35,36], spectral simulation was used to analyze some of the PWR data to provide insights into the structural consequences of peptide binding and aggregation Acknowledgements This work was supported, in part, by grants from the US National Institutes of Health (GM 59630 to G.T and Z.S.), from the Amgen Corporation (to G.T., Z.S and S.D.), from the Swedish Research Council (to ˚ G.L and G.G.), from the Umea University Biotechnology Fund (to G.G.) and from the Alzheimer Foundation (to G.G.) References Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL & Beyreuther K (1985) Amyloid plaque core protein in Alzheimer disease and Down syndrome Proc Natl Acad Sci USA 82, 4245–4249 Haass C & Selkoe DJ (1993) Cellular processing of beta-amyloid precursor protein and the genesis of amyloid beta-peptide Cell 75, 1039–1042 Rochet J-C & Lansbury PT Jr (2000) Amyloid fibrillogenesis: themes and variations Curr Opin Struct Biol 10, 60–68 Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, Taddei N, Ramponi G, Dobson CM & Stefani M (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases Nature 416, 507–511 Iversen LL, Mortishire-Smith RJ, Pollack SJ & Shearman MS (1995) The toxicity in vitro of beta-amyloid protein Biochem J 311, 1–16 Lansbury PT Jr (1999) Evolution of amyloid: what normal protein folding may tell us about fibrillogenesis and disease Proc Natl Acad Sci USA 96, 3342–3344 Walsh DM, Hartley DM, Kusumoto Y, Fezoui Y, Condron MM, Lomakin A, Benedek GB, Selkoe DJ & Teplow DB (1999) Amyloid beta-protein fibrillogenesis Structure and biological activity of protofibrillar intermediates J Biol Chem 274, 25945–25952 Walsh D, Tseng BP, Rydel RE, Podlisny MB & Selkoe DJ (2000) The oligomerization of amyloid beta-protein begins intracellularly in cells derived from human brain Biochemistry 39, 10831–10839 Klein WL, Stine WB Jr & Teplow DB (2004) Small assemblies of unmodified amyloid beta-protein are the FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS S Devanathan et al 10 11 12 13 14 15 16 17 18 19 20 21 22 23 proximate neurotoxin in Alzheimer’s disease Neurobiol Aging 25, 569–580 Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW & Glabe C (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis Science 300, 486–489 De Strooper B & Konig G (2001) An inammatory ă drug prospect Nature 414, 159–160 Yuan J & Yankner BA (2000) Apoptosis in the nervous system Nature 407, 802–809 Simons M, Keller P, De Strooper B, Beyreuther K, Dotti CG & Kimons K (1998) Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons Proc Natl Acad Sci USA 95, 6460–6464 Pettegrew JW, Panchalingam K, Hamilton RL & McClure RJ (2001) Brain membrane phospholipid alterations in Alzheimer’s disease Neurochem Res 26, 771–782 Kracun I, Kalanj S, Cosovic C, Talanhranilovic J & Cosovic C (1992) Cortical distribution of gangliosides in Alzheimer’s disease Neurochem Int 20, 433–438 Waschuk SA, Elton EA, Darabie AA, Fraser PE & McLaurin J (2001) Cellular membrane composition defines A beta–lipid interactions J Biol Chem 276, 33561–33568 McLaurin J, Yang D-S, Yip CM & Fraser PE (2000) Review: modulating factors in amyloid-beta fibril formation J Struct Biol 130, 259–270 Terzi E, Holzemann G & Seelig J (1997) Interaction of ă Alzheimer beta-amyloid peptide (140) with lipid membranes Biochemistry 36, 14845–14852 Bokvist M, Lindstrom F, Watts A & Grobner G (2004) ă ă Two types of Alzheimers beta-amyloid (140) peptide membrane interactions: aggregation preventing transmembrane anchoring versus accelerated surface fibril formation J Mol Biol 335, 1039–1049 Kakio A, Nishimoto S, Yanagisawa K, Kozutsumi Y & Matsuzaki K (2002) Interactions of amyloid beta-protein with various gangliosides in raft-like membranes: importance of GM1 ganglioside-bound form as an endogenous seed for Alzheimer amyloid Biochemistry 41, 7385–7390 Gibson Wood W, Eckert GP, Igbavboa U & Muller ă WE (2003) Amyloid betaprotein interactions with membranes and cholesterol: causes or casualties of Alzheimer’s disease Biochim Biophys Acta 1610, 281–290 Curtain CC, Ali FE, Smith DG, Bush AI, Masters CL & Barnham KJ (2003) Metal ions, pH, and cholesterol regulate the interactions of Alzheimer’s disease amyloidbeta peptide with membrane lipid J Biol Chem 278, 2977–2982 Kremer JJ, Pallitto MM, Sklansky DJ & Murphy RM (2000) Correlation of beta-amyloid aggregate size and hydrophobicity with decreased bilayer fluidity of model membranes Biochemistry 39, 10309–10318 Factors affecting Ab binding in lipid bilayers 24 Demeester N, Baier G, Enzinger C, Goethals M, Vandekerckhove J, Rosseneu M & Labeur C (2000) Apoptosis induced in neuronal cells by C-terminal amyloid beta-fragments is correlated with their aggregation properties in phospholipid membranes Mol Membr Biol 17, 219–228 25 Koppaka V & Axelsen PH (2000) Accelerated accumulation of amyloid beta proteins on oxidatively damaged lipid membranes Biochemistry 39, 10011–10016 26 Kawahara M, Kuroda Y, Arispe N & Rojas E (2000) Alzheimer’s beta-amyloid, human islet amylin, and prion protein fragment evoke intracellular free calcium elevations by a common mechanism in a hypothalamic GnRH neuronal cell line J Biol Chem 275, 14077– 14083 27 Yip CM & McLaurin J (2001) Amyloid-beta peptide assembly: a critical step in fibrillogenesis and membrane disruption Biophys J 80, 1359–1371 28 Yip CM, Darabie AA & McLaurin J (2002) Abeta42peptide assembly on lipid bilayers J Mol Biol 318, 97– 107 29 Kawarabayashi T, Shoji M, Younkin LH, Wen-Lang L, Dickson DW, Murakami T, Matsubara E, Abe K, Ashe KH & Younkin SG (2004) Dimeric amyloid beta protein rapidly accumulates in lipid rafts followed by apolipoprotein E and phosphorylated tau accumulation in the Tg2576 mouse model of Alzheimer’s disease J Neurosci 24, 3801–3809 30 Hooper NM (2005) Roles of proteolysis and lipid rafts in the processing of the amyloid precursor protein and prion protein Biochem Soc Trans 33, 335–338 31 Grobner G, Glaubitz C, Williamson PTF, Hadingham ă T & Watts A (2001) Structural insight into the interaction of amyloid-b peptide with biological membranes by solid state NMR In Perspectives on Solid State NMR in Biology (Kiihne SR & deGroot HJM, eds), pp 203–214 Kluwer Academic Publishers, Dordrecht 32 Lindstrom F, Bokvist M, Sparrman T & Grobner G ă ă (2002) Association of amyloid-b peptide with membrane surfaces monitored by solid state NMR Phys Chem Chem Phys 4, 5524–5530 33 Salamon Z, Macleod HA & Tollin G (1997) Coupled plasmon-waveguide resonators: a new spectroscopic tool for probing proteolipid film structure and properties Biophys J 73, 2791–2797 34 Salamon Z & Tollin G (2001) Plasmon resonance spectroscopy: probing molecular interactions at surfaces and interfaces Spectroscopy 15, 161–175 35 Salamon Z, Devanathan S, Alves I & Tollin G (2005) Plasmon-waveguide resonance studies of lateral segregation of lipids and proteins into microdomains (rafts) in solid-supported bilayers J Biol Chem 280, 11175–11184 36 Alves I, Salamon Z, Hruby V & Tollin G (2005) Ligand modulation of lateral segregation of a G-proteincoupled receptor into lipid microdomains in FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS 1401 Factors affecting Ab binding in lipid bilayers 37 38 39 40 41 42 S Devanathan et al sphingomyelin ⁄ phosphatidylcholine solid-supported bilayers Biochemistry 44, 9168–9178 Curtain CC, Ali F, Volitakis I, Cherny RA, Norton RS, Beyreuther K, Barrow CJ, Masters CL, Bush AI & Barnham KJ (2001) Alzheimer’s disease amyloid-beta binds copper and zinc to generate an allosterically ordered membrane-penetrating structure containing superoxide dismutase-like subunits J Biol Chem 276, 20466–20473 Bush AI (2003) The metallobiology of Alzheimer’s disease Trends Neurosci 26, 207–214 Lin H, Bhatia R & Ratneshwar L (2001) Amyloid beta protein forms ion channels: implications for Alzheimer’s disease pathophysiology FASEB J 15, 2433–2444 Ji S-R, Wu Y & Sui S-F (2002) Cholesterol is an important factor affecting the membrane insertion of beta-amyloid peptide (A beta 1–40), which may potentially inhibit the fibril formation J Biol Chem 277, 6273–6279 Bitan G, Kirkitadze MD, Lomakin A, Vollers SS, Benedek GB & Teplow DB (2003) Amyloid beta -protein (Abeta) assembly: Abeta 40 and Abeta 42 oligomerize through distinct pathways Proc Natl Acad Sci USA 100, 330–335 Micelli S, Meleleo D, Picciarelli V & Gallucci E (2004) Effect of sterols on beta-amyloid peptide (AbP 1–40) channel formation and their properties in planar lipid membranes Biophys J 86, 2231–2237 1402 43 Salamon Z, Brown MF & Tollin G (1999) Plasmon resonance spectroscopy: probing molecular interactions within membranes Trends Biochem Sci 24, 213–219 44 Tollin G, Salamon Z & Hruby VJ (2003) Techniques: plasmon-waveguide resonance (PWR) spectroscopy as a tool to study ligand–GPCR interactions Trends Pharmacol Sci 24, 655–659 45 Salamon Z, Wang Y, Tollin G & Macleod AH (1994) Assembly and molecular organization of self-assembled lipid bilayers on solid substrates monitored by surface plasmon resonance spectroscopy Biochim Biophys Acta 1195, 267–275 46 Salamon Z & Tollin G (2001) Optical anisotropy in lipid membranes: coupled plasmon-waveguide resonance measurements of molecular orientation, polarizability, and shape Biophys J 80, 1557–1567 47 Oradd G & Lindblom G (2004) Lateral diffusion stuă died by pulsed field gradient NMR on oriented lipid membranes Magn Reson Chem 42, 123–131 48 Oradd G, Westerman PW & Lindblom G (2005) Lateral ă diffusion coefcients of separate lipid species in a ternary raft-forming bilayer: a pfg-NMR multinuclear study Biophys J 89, 315–320 49 Ege C & Lee KY (2004) Insertion of Alzheimer’s A beta 40 peptide into lipid monolayers Biophys J 87, 1732– 1740 FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS ... measure the binding, insertion and aggregation of molecules, either at the lipid bilayer surface or upon incorporation into the bilayer The principles of creating a self-assembled single solid-supported. .. aggregation and neurotoxic action, in particular the presence of sphingomyelin, cholesterol and zinc ions Results and Discussion In order to characterize the interaction of Ab with lipid membranes,... organization of the membrane, including molecular anisotropy and the long range molecular order of the bilayer), and in protein ⁄ lipid conformation occurring as a consequence of molecular interactions