Tài liệu Báo cáo khoa học: Perturbation of membranes by the amyloid b-peptide – a molecular dynamics study pptx

Perturbation of membranes by the amyloid b-peptide – a molecular dynamics study Justin A Lemkul and David R Bevan Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA Keywords Alzheimer’s; amyloid; membrane; protein–lipid interactions; simulation Correspondence D R Bevan, Department of Biochemistry, Virginia Polytechnic Institute and State University, 201 Fralin Biotechnology Center, Blacksburg, VA 24061, USA Fax: +1 540 231 9070 Tel: +1 540 231 5040 E-mail: drbevan@vt.edu Website: http:// www.bevanlab.biochem.vt.edu (Received 19 February 2009, revised 24 March 2009, accepted 26 March 2009) doi:10.1111/j.1742-4658.2009.07024.x The etiology of Alzheimer’s disease is considered to be linked to interactions between amyloid b-peptide (Ab) and neural cell membranes Membrane disruption and increased ion conductance have been observed in vitro in the presence of Ab, and it is assumed that these same phenomena occur in the brain of an individual afflicted with Alzheimer’s The effects of Ab on lipid behavior have been characterized experimentally, but details are lacking regarding how Ab induces these effects Simulations of Ab in a bilayer environment can provide the resolution necessary to explain how the peptide interacts with the surrounding lipids In the present study, we present an extensive analysis of lipid parameters for a model dipalmitoylphosphatidylcholine bilayer in the presence of the 40-residue Ab peptide (Ab40) The simulated systems examine the effects of the insertion depth of the peptide, temperature, the protonation state of the peptide, and ionic strength on the features of the lipid bilayer The results show that Ab40 is capable of disordering nearby lipids, as well as decreasing bilayer thickness and area per lipid headgroup These phenomena arise as a result of the unfolding process of the peptide, which leads to a disordered, extended conformation that is capable of extensive electrostatic and hydrogen-bonding interactions between the peptide and the lipid headgroups Comparisons are made using melittin-dipalmitoylphosphatidylcholine systems as positive controls of a membrane-disrupting peptide because these systems have previously been characterized experimentally as well as in molecular dynamics simulations Alzheimer’s disease is a neurodegenerative disorder in which the hallmark symptoms include cognitive decline and dementia [1] Characteristic of this disorder is the formation of extracellular amyloid fibrils, and intracellular deposition of hyperphosphorylated tau [2] Alzheimer’s disease is considered to affect approximately five million Americans, and this number is expected to triple by the year 2050, according to recent estimates from the Alzheimer’s Association The number of Alzheimer’s patients worldwide has recently been estimated at 20–25 million [3] With the current annual health care costs estimated at $100 billion in the USA alone, the molecular basis for this disease is a topic of intense scientific research According to the ‘amyloid hypothesis’, interactions between the amyloid b-peptide (Ab) and other cellular components, especially membranes, are considered to give rise to the neurotoxicity observed in Alzheimer’s disease Ab is derived from the amyloid precursor protein by sequential proteolytic cleavage by two membrane-bound proteases, b- and c-secretase [2] The length of the peptide is variable, ranging from 39 Abbreviations Ab, amyloid b-peptide; Ab40, 40-residue alloform of the amyloid b-peptide; DMPC, dimyristoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; MD, molecular dynamics 3060 FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS J A Lemkul and D R Bevan to 43 amino acids, with the 40- and 42-residue alloforms being the most common The peptide is considered to be partially embedded in the cell membrane [4], but it can exit over time and accumulate in the extracellular environment, giving rise to the neuritic plaques observed in the brains of Alzheimer’s patients Because Ab is localized in the plasma membrane, an analysis of the interactions between the peptide and the membrane environment is crucial to understanding the exit pathway of the peptide and the manner in which it disrupts membranes There are two proposed positions for Ab within the plasma membrane, as determined by experiments performed in vitro: one with Val24 at the membrane–water interface, the other with Lys28 at the interface [4,5] The location of the peptide within the membrane may affect the types of interactions that it has with the surrounding lipid matrix and the pathway that it follows to exit from this environment The use of atomic-force microscopy has concluded that the 40-residue form of Ab (Ab40) is partially embedded in model dimyristoylphosphatidylcholine (DMPC) micelles [6] Work conducted in vitro with Ab in the presence of rat synaptic plasma membranes has shown that monomeric Ab can intercalate into the bilayer interior and lead to decreased bilayer thickness [7] The same study also concluded that Ab40 increased the fluidity of the lipids in the membrane, in agreement with a previous study [8] However, the effects of Ab on lipid fluidity are contentious because another study found that Ab decreased the fluidity of the surrounding lipids [9] In disturbing the integrity of the plasma membrane, Ab promotes the increased leakage of ions, particularly calcium, into the cell [10] The disruption of calcium homeostasis, and thus the promotion of neuronal excitotoxicity, is considered to be a component of Alzheimer’s disease Perturbation of the plasma membrane in the presence of Ab has been noted in several studies [11,12] Although a study by Kayed et al [11] concluded that permeabilization of the plasma membrane was only caused by oligomeric Ab, it was also noted that monomeric and low-molecular weight Ab species could incorporate into the membrane and cause a reduction in the thickness of the bilayer, and this observation was corroborated by Ambroggio et al [12] These investigators found that Ab42 could stably incorporate into the plasma membrane and reduce the cohesive forces between surrounding lipids Perturbation of membranes has been associated with other toxic peptides and proteins, most notably melittin, a component of bee venom that is considered to exert its toxic effect by associating with cell Membrane perturbation by Alzheimer’s Ab membranes [13–15] Model systems of melittin in dipalmitoylphosphatidylcholine (DPPC) and DMPC bilayers have been studied by molecular dynamics (MD) simulations [16–18], demonstrating that melittin interacts asymmetrically with the leaflets of the bilayer and can draw water into the membrane That is, the peptide disorders the leaflet with which it interacts most closely (i.e the extracellular face), at the same time as increasing lipid order in the cytofacial leaflet Simulations of melittin in DPPC lead to an interesting comparison with the Ab-DPPC simulations reported in the present study Both melittin and Ab are short, mostly helical peptides that are assumed to be asymmetrically oriented with respect to the membrane Both are considered to cause some amount of disorder on the surrounding lipid environment Because the interactions between melittin and lipids have been well-characterized in previous MD simulations, we used melittin–membrane systems as a basis for interpreting the disruptive effect of Ab40 on its surrounding lipid environment The success of applying MD to membrane protein systems has been well documented, and simulations have illustrated the conformational dynamics of proteins embedded in membranes [19,20] as well as the interactions between proteins and the surrounding lipids [19,21,22] A recent review has discussed these phenomena in detail [23], highlighting many of the parameters that have been successfully measured in membrane protein MD simulations To our knowledge, only three studies have examined Ab in an explicit bilayer environment [24–26], but none of these have reported the behavior of the lipid membrane in which the peptide was embedded and, instead, have focused primarily on the properties of the peptide In the present study, we aimed to expand previous work by examining the properties of lipid molecules surrounding the membrane-perturbing Ab40 peptide Although it is known that Ab can interact with the plasma membrane and assemble in this environment [6], a fundamental understanding of the molecular basis for this phenomenon is missing Central questions still remain, especially regarding the intrinsic characteristics (i.e both structural and chemical) of Ab that allow it to disrupt the surrounding lipids Detailed studies with atomic resolution, such as the simulations reported in the present study, are crucial to understanding this phenomenon A greater knowledge of the most basic interactions between Ab and a model membrane can lead to a more complete understanding of the membrane-aided assembly of Ab and the resulting damage to cell membranes FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS 3061 Membrane perturbation by Alzheimer’s Ab J A Lemkul and D R Bevan Results Table DPPC control simulation details System Ionic strength (mM) OS1 OS2 NS1 NS2 NS3 0a 100 100 Description of simulation systems The preparation of the ten Ab-DPPC simulation systems listed in Table has been described in detail elsewhere [25] and only a summary of their essential characteristics is appropriate here The coordinates and topology for the DPPC bilayer were obtained from a previous study by Tieleman and Berendsen [27], and are available at the author’s website (http:// moose.bio.ucalgary.ca/index.php?page=Structures_and_ Topologies) The goals of these simulations were to examine the effects of different variables (i.e ionic strength, temperature, and Ab positioning) on the dynamics of the peptide and the behavior of the surrounding lipids Systems belonging to simulation set A were designed primarily to understand the effects of an increased salt concentration Simulation set B examined the effects of both the protonation state of the peptide and temperature on the behavior of the system Finally, the systems in simulation set C also examined the effects of increased salt, but contrasted with simulation set A in that the Ab peptide was placed more deeply in the membrane Two sets of negative control systems of pure DPPC bilayers were prepared by a similar method These systems were designed to examine whether the additional solvation or increased ionic strength had any background effect on lipid dynamics Two systems were prepared from this structure: (a) the original bilayer with the original water-to-lipid solvation ratio (‘Original Solvation’; OS1) and (b) this bilayer in the presence of 100 mm NaCl (OS2) In addition, three systems were prepared by placing an additional slab of water to one side of the bilayer to approximate the Table Simulation system details Ionic Net strength charge DPPC Temperature Water Solvation System (mM) on Ab lipids (K) molecules ratio A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 a 0a 100 100 0 0 100 )3 122 323 122 300 323 300 323 323 )2 +1 +7 )3 120 6912 6873 6922 6878 6949 6948 6951 6948 6928 6888 56.6 56.3 56.7 56.4 57.0 57.0 57.0 57.0 57.7 57.4 : : : : : : : : : : 1 1 1 1 1 An ionic strength of mM implies counterions sufficient only to neutralize the charge of the system 3062 DPPC lipids Temperature (K) 128 323 128 300 Water molecules Solvation ratio 3655 3641 6907 6881 6907 28.6 28.4 54.0 53.8 54.0 : : : : : 1 1 a An ionic strength of mM implies that no ions were added to these systems increased water-to-lipid solvation ratio and the system size present in the peptide–bilayer systems Similar to the OS simulation set, these systems contained either no salt (‘New Solvation’; NS1 and NS3) or 100 mm NaCl (NS2) The solvation ratios of NS1, NS2, and NS3 closely match those of the simulated Ab systems, although not exactly Instead, they were designed to strike a balance between the dimensions of the system and the number of water molecules aiming to examine whether or not the asymmetry of the system and increased solvation would affect the dynamics of the lipids System details are summarized in Table In each simulation, coordinates were saved every ps, generating 50 000 data points per simulation Analyses were conducted using tools within the gromacs software package, version 3.3 [28] (for deuterium order parameters) and code developed in-house [29] (for lipid tilt, effective chain length, area per lipid headgroups, and bilayer thickness) Averaging over time was conducted, when appropriate, to generate a time-dependent progression of these measurements Positive control systems were prepared with melittin the presence of DPPC The structure of melittin was taken from the crystal structure, Protein Data Bank entry 2MLT [30] Two orientations were prepared: one with melittin embedded in the DPPC bilayer, as in previous studies [17,18] [‘Embedded’ (E1) and with 100 mm NaCl (E2)], and the other with melittin parallel to the bilayer interface, as reported previously [16] [‘Parallel’ (P1) and with 100 mm NaCl (P2)] These systems were prepared in the same manner as the Ab-DPPC systems, giving starting configurations comparable to those presented in the original studies Details of these systems are presented in Table The initial asymmetric orientation of Ab relative to the DPPC bilayer creates an interesting situation when analyzing the properties of the surrounding lipid bilayer Over time, the peptide interacts differently with each leaflet Such a situation resembles that of melittin, whose interactions with lipids have been FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS J A Lemkul and D R Bevan Membrane perturbation by Alzheimer’s Ab  Table Melittin simulation details ÀSCD ¼ System Ionic strength (mM) E1 E2 P1 P2 0a 100 100 DPPC lipids Temperature (K) Water molecules 323 118 323 7071 6993 7047 7005 59.4 58.8 59.7 59.4  ð1Þ Solvation ratio 119 cos2 h À : : : : 1 1 a An ionic strength of mM implies counterions sufficient only to neutralize the charge of the system described experimentally [31–34] and computationally [16–18] Validity of melittin-DPPC controls relative to previous work ` Studies by Bachar and Becker [18] and Berneche et al [16] provide meaningful reference points to the simulations of the present study We constructed systems that had initial configurations similar to those produced by the original investigators, but we simulated the systems in a different manner in certain respects We applied new equilibration schemes to pack the lipids around the peptides, using different force field parameters (GROMOS ⁄ Berger instead of CHARMM) We also conducted simulations that were far longer than in the original reports (i.e 100 ns instead of 300–500 ps) The goal of this series of simulations was to produce data not only to validate our simulation set-up, but also to serve as a basis for comparing the effects of Ab40 on a DPPC bilayer in light of the observations made with respect to melittin Simulation E1 was inspired by the work of Bachar and Becker [18], and simulation E2 arose from our desire to examine the effects of increased ionic strength on the peptide–membrane systems Even in our longer 100 ns simulations, the positioning and orientation of melittin at the end of the simulations were similar to that reported by Bachar and Becker [18], although, in our simulations, melittin became embedded more deeply in the bilayer and more disordered at its termini The disordering at the termini was predicted by Bachar and Becker [18], although it was not observed in the timeframe that they simulated With respect to the lipid properties, the most meaningful comparison between the present study and previous simulations arises with respect to lipid order Deuterium order parameters describe the orientation of the lipid acyl chains, on average, relative to the bilayer normal These parameters are calculated by the equation: In Eqn (1), h represents the angle between the C-D bond and the bilayer normal, and the angle brackets denote that the values are averaged over all equivalent atoms, and over time We observed that the lipids nearest melittin experience a greater degree of disorder, whereas more distant lipids become more ordered relative to control simulations in the absence of melittin This disordering effect is comparable to the results obtained in the original studies [16,18] In addition, the top leaflet of the bilayer, which interacts with melittin most strongly, was observed to be more disordered relative to the bottom leaflet, which experienced a greater degree of chain elongation and lipid packing Bachar and Becker [18] divided the lipids in their bilayer into ‘tiers’ based on the distance between the protein and lipid molecule center of mass The average value of )SCD was presented for the ‘plateau region’ of the acyl chain, which extends from carbons to of the acyl chain (denoted [4,8]) The values reported are 0.157 ± 0.009, 0.215 ± 0.006, and 0.215 ± 0.006 for the first, second, and third tiers, respectively We find very similar values of 0.144 ± 0.010, 0.194 ± 0.007, and 0.219 ± 0.005 for these same subsets of lipids We attribute the small differences in these values to the use of different force fields, application of different equilibration schemes, and the length of our simulations, which is several orders of magnitude longer than that of the original study Similar conclusions can be made between our simulation P1 (i.e starting with melittin parallel to the interface of the bilayer at the beginning of the simulation) ` and the study by Berneche et al [16] With respect to the behavior of the lipids, we make the same observation that those of the top leaflet (which also interact most strongly with melittin) become very disordered relative to the lipids of the lower leaflet, overall, although our values for the deuterium order parameters are ` higher In the original study by Berneche et al [16], the average order parameter was 0.149 in the top leaflet and 0.188 (i.e a difference of 21%) in the bottom leaflet The corresponding values for these parameters from our simulations are 0.157 and 0.220 (29% difference), respectively We attribute these differences to many of the same factors as described above with respect to the study by Bachar and Becker [18], and also the fact that ` the simulations conducted by Berneche et al [16] utilized DMPC as the membrane lipid instead of DPPC, so that some differences should be expected FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS 3063 Membrane perturbation by Alzheimer’s Ab J A Lemkul and D R Bevan Deuterium order parameters of the Ab40-DPPC systems In our studies with Ab40, we examine a peptide that is primarily asymmetric with respect to its interactions with the lipid membrane As such, we analyzed the deuterium order parameters of each leaflet separately, including the whole acyl chain and the ‘plateau region’ described above Taking the approach applied by Bachar and Becker [18], we analyzed the lipids in ‘tiers’ at increasing distance from Ab40 The first and second tiers contained 20 lipids each, and the third tier contained the remaining lipids in the leaflet, between 18 and 24 The results of these calculations are presented in Table Note that the tiered analysis does not apply to the DPPC-only controls; the values presented represent an average of the plateau region for each leaflet of the bilayer From these data, it can be seen that, overall, the )SCD values in the top leaflet of the Ab40-DPPC systems are lower than those in the bottom leaflet One explanation for this phenomenon was proposed by Tieleman et al [35], wherein the lipids that interact strongly with the protein become increasingly tilted relative to the bilayer normal, causing the angle between the C-D bond and the normal to decrease This occurrence was reported in simulations of melittin [18], and occurs in the present study as well in the case of both melittin and Ab40 The lipids of the top leaflet tend to adopt an angle such that they become tilted, with their headgroups pointing towards Ab, and the lipids of the lower leaflet elongate to become more ordered, filling the void in the center of the bilayer (Fig 1) The results of simulations C1 and C2 reflect the fact that the peptides in these simulations interacted more or less symmetrically with both leaflets over time The peptide became deeply inserted in the bilayer in a transmembrane orientation, with disordered N- and C-termini protruding through the lipid headgroups of both leaflets We also note that the lipids in the first tier tend to be more disordered than those of the second and third tiers In fact, in most cases, the values of [4,8] increase as the distance between the peptide and the lipids increases The presence of the Ab40 peptide causes substantial disorder on the lipids with which it most closely interacts, simultaneously resulting in an increase in order of the lipids that are further away This behavior is dependent upon the conformation of the peptide In cases where Ab40 lost much of its initial a-helicity, the nearby lipids become more disordered and the more distant lipids increase in order In cases where the peptide unfolds to a lesser extent (e.g simulation B4), the distant lipids approach a value of [4,8] that is comparable to that of the relevant control (NS1), based on the average order parameter of Tier in the two leaflets We thus conclude from these data that Ab interacts with the membrane in a Table Average values of deuterium order parameters Data are the mean (± SD) Top leaflet plateau region Simulation Tier A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 OS1 OS2 NS1 NS2 NS3 E1 E2 P1 P2 0.163 0.209 0.147 0.188 0.205 0.134 0.233 0.160 0.161 0.183 (0.007) (0.008) (0.008) (0.009) (0.019) (0.008) (0.007) (0.010) (0.010) (0.012) 0.166 0.217 0.177 0.202 (0.007) (0.005) (0.013) (0.009) 3064 Tier 0.223 0.274 0.219 0.250 0.243 0.213 0.249 0.220 0.240 0.284 0.216 0.232 0.217 0.242 0.252 0.218 0.252 0.200 0.228 Bottom leaflet plateau region Tier (0.008) (0.006) (0.005) (0.004) (0.016) (0.006) (0.013) (0.006) (0.005) (0.007) (0.004) (0.004) (0.003) (0.004) (0.007) (0.004) (0.005) (0.005) (0.004) Tier Tier 0.233 0.285 0.231 0.273 0.318 0.227 0.307 0.239 0.288 0.321 (0.007) (0.011) (0.004) (0.006) (0.017) (0.004) (0.014) (0.008) (0.007) (0.012) 0.180 0.254 0.192 0.197 0.189 0.165 0.212 0.220 0.200 0.232 (0.005) (0.010) (0.006) (0.015) (0.019) (0.004) (0.008) (0.006) (0.008) (0.010) 0.235 0.245 0.203 0.247 (0.005) (0.005) (0.004) (0.006) 0.206 0.225 0.176 0.205 (0.010) (0.004) (0.012) (0.007) 0.211 0.235 0.191 0.247 0.271 0.193 0.246 0.191 0.221 0.269 0.215 0.231 0.217 0.243 0.253 0.193 0.253 0.205 0.229 Whole acyl chain Tier (0.009) (0.006) (0.005) (0.006) (0.020) (0.006) (0.014) (0.004) (0.005) (0.005) (0.004) (0.005) (0.004) (0.003) (0.009) (0.004) (0.008) (0.008) (0.006) Top 0.216 0.263 0.203 0.250 0.268 0.203 0.286 0.197 0.247 0.275 (0.006) (0.010) (0.009) (0.003) (0.018) (0.006) (0.010) (0.007) (0.006) (0.009) 0.211 0.232 0.198 0.228 (0.005) (0.007) (0.005) (0.004) Bottom 0.177 0.223 0.173 0.207 0.231 0.167 0.240 0.176 0.204 0.240 0.185 0.201 0.188 0.213 0.229 0.178 0.210 0.157 0.187 0.217 0.260 0.231 0.238 0.263 0.213 0.280 0.213 0.196 0.245 0.185 0.201 0.188 0.214 0.229 0.206 0.245 0.220 0.252 FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS J A Lemkul and D R Bevan Membrane perturbation by Alzheimer’s Ab peptide–membrane systems is not substantially different from that of the control (DPPC-only) systems (see Fig S1) Bilayer thickness It has been reported previously that monomeric Ab40 can intercalate into the hydrophobic core of reconstituted synaptic plasma membranes, resulting in a decrease in the thickness of the membrane [7] To quantitatively assess this descriptor of membrane disruption, we measured the thickness of our simulated bilayers in terms of the P–P distance between the top and bottom leaflets of the bilayer, using gridmat-md [29] The results obtained are shown in Fig More detailed results are provided in the Supporting information (Figs S2–S6) The time averages over the last Fig As shown in a snapshot from the end of trajectory A1, the Ab40 peptide causes DPPC lipids of the top leaflet of the bilayer to become more disordered, with their acyl chains becoming more parallel to the bilayer surface Lipids in the bottom leaflet become more ordered, extending their acyl chains to fill in the growing void in the center of the bilayer Representative lipids (sticks) near the peptide (ribbon) are shown manner similar to melittin with respect to its effects on the disordering of the surrounding lipids In addition, the )SCD values for the top leaflet lipids of the peptide–membrane systems are primarily lower than the respective controls (NS1 or NS2), whereas the bottom leaflet lipids are more ordered than the controls This behavior is a result of the top leaflet interacting strongly with the unfolded and charged portions of the peptides in each simulation, and is especially true in the case of the lipids closest to the peptide (Tier 1) The values of )SCD for the controls are in good agreement with previous experimental and simulation studies [36,37] The contraction of the lipid headgroups, and concomitant disordering of the acyl chains of the lipids in closest contact with Ab, results in no substantial changes in the overall density of the lipid bilayer There is a slight increase in density among the lipids nearest Ab (most likely a result of the strong interaction between Ab and the lipid headgroups; see below), but regions of slightly lower density exist to compensate for this more tightly-packed region The bottom leaflet, which becomes more ordered over time, increases in density slightly The top leaflet appears to be slightly less dense than the bottom leaflet as well as the control Factoring in the presence of the protein and averaging between the two leaflets gives an overall result that the bulk density of the lipids in the Fig Bilayer thickness around the embedded peptides, taken from the average thickness over the last 25 ns of simulation Peptide conformations are from the final frame of each simulation, which is representative of the final 50 ns of simulation time For perspective, the embedded region of the peptide is colored gray, whereas the region exposed to the water–bilayer interface is shown in black The legend shows bilayer thickness (nm), mapped to the corresponding colors FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS 3065 Membrane perturbation by Alzheimer’s Ab J A Lemkul and D R Bevan 25 ns of each trajectory are shown The final conformation of the peptide is also shown, placed at its average location over this time period The final conformation of the peptide is representative of the last 50 ns of the trajectories because most of the prominent secondary structure changes occurred during the first half of each simulation [25] The most striking observation overall is the amount by which the Ab40 peptide depresses the bilayer in its immediate vicinity, in the order of 1.0 nm There are hydrogen bonds and favorable electrostatic interactions between the zwitterionic headgroups of the DPPC lipids and the backbone and charged residues of the peptide The result of these interactions is that the lipid headgroups tilt substantially around the peptide, causing the acyl chains of the lipids to spread outward, more parallel to the surface of the bilayer (Fig 1; see below) Control simulations above the phase transition temperature (i.e those without embedded peptides, at 323 K) show good agreement with the experimentally-determined thickness of 3.7 nm [38] It is also observed that melittin can lead to a similar magnitude of bilayer thinning, in the order of 0.5–1.0 nm This thinning only occurs in regions where the peptide became more disordered over time For simulations E1 and E2, these disordered segments were the N- and C-termini of the peptide, whereas it was the N-terminus in P1, and the middle of the peptide became slightly disordered in P2 Area per lipid headgroup Experimental work has concluded that the average area per lipid headgroup for fully hydrated DPPC at ˚ 50 °C is in the range 62–64 A2 [39,40] Previous simulations of DPPC examining the effects of increased ionic strength have demonstrated that the area per lipid headgroup decreases with an increasing salt con˚ centration, from 62.7 A2 in the absence of NaCl to ˚ in the presence of 100 mm NaCl [41] The 60.5 A results from our control systems, averaged over the last 50 ns of simulation (Table 5), compare well with these findings There is very little difference between DPPC systems at the original solvation ratio, and those in an asymmetric box with an increased amount of water (NS1, NS2, and NS3) Determining the area per lipid headgroup in the presence of an irregularly-shaped protein presents a unique challenge, and we utilize the gridmat-md methodology, wherein each headgroup is assigned to a polygon within the grid of the lateral bilayer surface [29] As shown in Table 5, a trend becomes clear The area per lipid headgroup for lipids in the top leaflet is decreased substantially from the control simulations, whereas the area per lipid headgroup for lipids in the ˚ Table Area per lipid headgroup (mean ± SD) in A2 (% difference from controls) over the last 50 ns of each trajectory Simulation A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 OS1 OS2 NS1 NS2 NS3 E1 E2 P1 P2 Residue initially at bilayer–water interface Simulated temperature (K) K28 323 K28 300 323 300 323 323 V24 NA W19 NA 323 323 323 323 300 323 Top leaflet 52.7 49.0 54.4 49.0 46.8 55.9 48.0 52.9 53.6 49.7 63.3 60.5 63.5 59.3 58.7 58.7 56.0 62.5 57.1 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.4 1.7 1.0 0.8 0.6 1.3 0.9 1.4 1.4 1.5 0.7 1.0 1.1 0.7 0.7 1.1 0.8 1.8 1.1 Bottom leaflet ()17%) ()17%) ()14%) ()17%) ()20%) ()12%) ()18%) ()17%) ()16%) ()16%) ()7.6%) ()5.6%) ()1.6%) ()3.7%) 60.0 54.5 58.5 57.4 55.2 61.6 54.1 60.4 61.7 55.9 63.3 60.5 63.5 59.3 58.7 59.5 55.4 60.7 55.7 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.8 1.1 1.0 1.0 0.5 0.8 0.8 0.8 1.6 1.4 0.7 1.0 1.1 0.7 0.7 1.1 1.3 0.9 0.8 ()5.5%) ()8.1%) ()7.9%) ()3.2%) ()6.0%) ()3.0%) ()7.8%) ()4.9%) ()2.8%) ()5.7%) ()6.3%) ()6.6%) ()4.4%) ()6.0%) In the case of the OS ⁄ NS series, no peptide was present For P1 and P2, the entire peptide was initially located at the membrane–water interface NA, not applicable 3066 FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS J A Lemkul and D R Bevan bottom leaflet is only slightly less than that of the controls The reason for this result is related to the observations regarding deuterium order parameters The lipids of the top leaflet interact strongly with the backbone and charged amino acid side chains of the disordered N-terminal segment of Ab via hydrogen bonds and electrostatic interactions The result is that the lipids tilt substantially, reducing the vertical thickness of the top leaflet This behavior requires the lipids of the lower leaflet to pack more tightly and extend their acyl chains to maintain the integrity of the membrane The substantial tilt (and resulting disorder) of the top leaflet lipids and the slight increase in packing (and thus order) in the bottom leaflet lipids is reflected in the area per lipid headgroup The interaction between the N-terminal segment of Ab and the DPPC headgroups develops over time After equilibration, the measured area per lipid headgroup in each system is close to the accepted experi˚ mental value (62–64 A2) Upon contact between the Membrane perturbation by Alzheimer’s Ab N-terminal region of Ab and the membrane–water interface (within 10 ns of simulated time), the area per lipid headgroup begins to rapidly decrease as the lipids associate with this disordered segment of the peptide (Fig 3; see also Figs S7–S9) Unfolding of Ab occurs over the first 50 ns of each simulation, after which the peptide conformation is largely unchanged [25] The area per lipid headgroup for the control systems (simulation sets OS and NS) remains steady over time at values appropriate for a fully hydrated DPPC bilayer under the given conditions (Fig 4) The lipids closest to Ab40 experience the greatest decrease in area per lipid headgroup From Fig 5, it can be seen that lipids closest to the peptide have the smallest lateral area, whereas lipids further away tend to occupy areas close to the bulk value of DPPC In Fig 5, lipids of the top leaflet were ordered according to their proximity to the center of mass of the Ab peptide Thus, the closer lipids have the smaller residue Fig Area per lipid headgroup as a function of time for simulation set A After making contact with the DPPC headgroups (within 10 ns in all cases), the N-terminal segment of Ab attracts the lipids of the top leaflet, depressing their lateral area The area per lipid headgroup in the bottom leaflet is decreased as a result of the increased order and packing in this leaflet, which is a consequence of the disordering of the top leaflet FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS 3067 Membrane perturbation by Alzheimer’s Ab J A Lemkul and D R Bevan Fig Area per lipid headgroup as a function of time for control DPPC simulations Fig Area per lipid headgroup as a function of distance from the protein; simulations A1 and A2 are shown at each of three time points (0, 50, and 100 ns) Lipid residues are numbered such that those closest to the peptide have the lowest numbering, increasing as the lipids are further away from the peptide For clarity, running averages of the data are shown, using a window of ten data points designation In the case of simulation A1, the area per lipid headgroup is largely constant at the outset of the ˚ simulation, fluctuating around a value of 62 A2 Over time, the lipids close to the peptide are drawn to it by the interactions described above, whereas more distant lipids maintain a more canonical value for their lateral 3068 area This trend is apparent in all other simulations of Ab (see Figs S10–S15), except for A2 In simulation A2, the peptide unfolded to the greatest extent of any of the simulations, thus contacting the greatest number of lipids The lipids closest to the peptide center of mass have a depressed value for their lateral area, as FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS J A Lemkul and D R Bevan Membrane perturbation by Alzheimer’s Ab its secondary structure over time, the effects of the peptide on this parameter are less pronounced than in the case of Ab In simulations P1 and P2, wherein the entire peptide was in contact with the DPPC headgroups, the nearest lipids experienced a reduction in their lateral area, which we attribute to hydrogenbonding between charged headgroup phosphates and the backbone of the small section of the peptide that became disordered over time (Fig 2; see also Fig S15) Lipid tilt and effective chain length Fig Illustration of the contacts between the Ab40 peptide in simulation A2 and the lipids of the top leaflet The peptide is shown as a black ribbon, and each lipid is represented by the phosphorus of its headgroup, shown as spheres The phosphorus atoms are colored according to the lateral area of the corresponding lipid, increasing as the colors change from blue to red The small green sphere represents the peptide center of mass, demonstrating that not all of the lipids closest to this point experience the greatest degree of association with the peptide in this simulation some lipids that are more distant from this point The explanation for this behavior is that, as the disordered N-terminal segment elongates through the lipid headgroups, it interacts with a greater number of lipids than in any other simulation In Fig 5, lipids numbered from 41 to 49 are close to the peptide center of mass and are attracted by contact with the polar backbone, lipid residues 50–60 make van der Waals contacts with the amino acid side chains of Ab, and lipid residues 60–70 interact strongly with the highly charged and disordered N-terminal segment of Ab (Fig 6) In all cases, the area per lipid headgroup in the bottom leaflet was largely insensitive to proximity to the peptide, even in the simulations wherein the C-terminus of Ab interacted with the lipids of the lower leaflet (A3, C1, and C2) This observation indicates that the ability of Ab to condense nearby lipids lies primarily in its highly-charged, unstructured N-terminal segment Simulations of melittin showed similar behavior Simulations E1 and E2 showed a slight decrease in area per lipid headgroup in the vicinity of the peptide (see Fig S13) Because melittin largely maintains The attraction between the lipids and unfolded regions of the Ab peptide described above gives rise to the striking behavior of the lipid acyl chains As noted above, the acyl chains of lipids near the peptide tilt substantially, increasing their disorder as the peptide draws them close to itself To quantify this observation, two related parameters were measured: acyl chain tilt angle and effective chain length We defined the acyl chain tilt angle as the angle formed between the bilayer normal and the vector defined by the first methylene carbon and the terminal methyl carbon on the acyl chain A description of effective chain length has been proposed by Petrache et al [42] (therein termed the ‘average chain length’; LC*) This descriptor is simply defined as the distance along the bilayer normal between the first methylene carbon and the terminal methyl carbon These two parameters (i.e the tilt angle and the effective chain length) should be related under most circumstances, such that, as the tilt angle increases (and the acyl chain becomes more parallel to the bilayer surface), the effective chain length should decrease Tilt angle and effective chain length have been analyzed for the systems simulated in the present study as a function of distance from the peptide There was no substantial difference in the results for the sn-1 and sn-2 chains; hence, for the purpose of clarity, the data presented here are in direct reference to the sn-1 chain We find that the lipids in the top leaflet in closest contact with the peptide (typically those interacting with the disordered N-terminal segment) increase their tilt angle over time, simultaneously decreasing their effective chain length (Fig 7) In other words, the strong attraction between the peptide backbone and charged residues draws the headgroups of nearby lipids away from other surrounding lipids, pulling the entire lipid more parallel to the surface of the bilayer Regions of the most substantially tilted lipids correspond to those with the smallest area per lipid headgroup and the greatest amount of disorder In the bottom leaflet, the FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS 3069 Membrane perturbation by Alzheimer’s Ab J A Lemkul and D R Bevan Fig Effective chain length (top panel) and acyl chain tilt angle (bottom panel), as a function of distance from the protein, for simulation A1 The results are indicative of all the other simulations involving Ab, and thus are representative of the general conclusions discussed in the text There is no substantial difference in the characteristics of the sn-1 and sn-2 chains; thus, only the results for the sn-1 chain are presented for clarity lipid chains elongate, as demonstrated by a small increase in effective chain length over time, as well as an overall reduction in the tilt angle (Fig 7) Lipid tilting was minimal in simulations involving melittin, which interacts more weakly with the lipid headgroups as a result of its smaller size and greater retention of secondary structure These results demonstrate that more extensive lipid tilting is induced by the dynamic behavior of Ab Because Ab unfolds to a much greater extent than melittin, thus interacting with more lipids, it is able to cause greater disruption of canonical lipid dynamics and orientation Control simulations of pure DPPC showed an effective chain length of approximately 1.25 nm in both leaflets, which is in agreement with the value proposed by Petrache et al [42] Discussion According to the ‘amyloid hypothesis’, Ab is central to the development and progression of Alzheimer’s disease 3070 [2], but relatively little is known about how this small peptide interacts with a lipid membrane in the context of neurodegeneration Although a number of experiments have examined the properties of lipids in the presence of Ab [7,11,12], little detailed structural data exist to indicate how Ab induces these observed phenomena, providing the motivation for the present study To gain a clear picture of how Ab disrupts a membrane environment, the peptide must be examined in detail with respect to its structural and chemical features and how they impact the surrounding lipid matrix A previous study by Ambroggio et al [12] suggested that Ab42 interacts strongly with a lipid environment, becoming part of it and disrupting interactions between the lipids, leading to structural deformation Although the focus of their study was Ab42, whereas Ab40 was the subject of investigation in the present study, we consider that there are common features of both alloforms contributing to membrane perturbation The most substantial observations of the present study are that the Ab40 peptide causes nearby DPPC FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS J A Lemkul and D R Bevan lipids to become more tilted and disordered relative to controls, that the peptide is capable of reducing the area per lipid headgroup of the lipids with which it most directly interacts, and that it is capable of reducing the thickness of the membrane in its immediate vicinity Taken as a whole, these data suggest interesting roles for the region of the peptide that is present in the extracellular environment, and that which remains embedded in the bilayer Lipid tilt and effective chain length The factor that gives rise to much of the behavior discussed in the present study is the tilting of lipids that are closest to the peptide present in each simulation The Ab peptide is capable of drawing lipid headgroups to itself through electrostatic and hydrogen-bonding interactions, weakening the interactions between these lipids and others that are more distant from Ab Nearby acyl chains tilt substantially over time (Fig 7), leading to a slight thinning of the hydrophobic core of the bilayer, which is manifested in a reduction in effective chain length of these same lipids Lipids that displayed the greatest degree of tilting also correspond to those with the smallest area per lipid headgroup and the greatest amount of disorder We attribute these observations to the ability of the Ab40 peptide, especially through its N-terminal disordered region, to bind lipid headgroups very closely to itself and draw them closely to each other Further details of this phenomenon are provided below, where the effects of lipid tilting on each of the other parameters measured in the present study are described Area per lipid headgroup We previously described the unfolding of the Ab40 peptide in these simulations [25], and noted that, in each simulation, the extracellular region of the peptide (typically residues 1–28) interacted strongly with the water–bilayer interface region and became disordered in almost all simulations We attributed this behavior to strong hydrogen-bonding and electrostatic interactions between the peptide and the zwitterionic phosphatidylcholine headgroups This behavior creates an interesting contrast with melittin, another membranedisrupting peptide When embedded, melittin still has a small portion of its structure exposed to the extracellular environment, but it is substantially shorter than the extracellular segment of Ab40, and thus retains a more organized secondary structure than Ab40 in the simulations conducted in the present study Table shows that the effect of melittin on the area per lipid Membrane perturbation by Alzheimer’s Ab headgroup of the individual bilayer leaflets is minimal, even when the whole peptide is positioned approximately parallel to the water–bilayer interface There is some reduction in the area per lipid headgroup by melittin, but the difference in area per lipid headgroup between the leaflets is almost indistinguishable However, in the case of Ab40, there is often a prominent difference in the area per lipid headgroup between the leaflets, with the leaflet interacting with the extracellular (N-terminal) region of Ab40 experiencing a substantially reduced area per lipid headgroup relative to the intracellular leaflet This is true even in the case of simulations A3, C1, and C2, in which the peptide adopted a transmembrane orientation, interacting with the lipid headgroups in the bottom leaflet as well We propose that this behavior arises because of different interactions between the peptides and the lipid headgroups In the case of Ab40, the peptide is capable of attracting lipid headgroups very close to its long, mostly disordered, N-terminal segment, tilting the lipids and arranging them very closely to each other This finding is independent of the protonation state and ionic strength of the surrounding aqueous medium, suggesting that electrostatic interactions and hydrogen-bonding are likely to be involved in drawing lipid headgroups in, but that these interactions are nonspecific In other words, they are not sensitive to the protonation state of any particular residue or group of residues They occur simply because there are many charged and polar amino acids in the extracellular region of Ab40, which interacts with the elements of the water–bilayer interface Although some lipids become oriented such that their headgroups interact with the C-terminal region of melittin, they tend to remain more dispersed compared to the lipids in the Ab40 simulations That is, there are fewer lipids tightly associated with melittin than there are in the case of Ab40 Disordering of nearby lipids In our simulations, both Ab40 and melittin demonstrated the ability to cause disorder in nearby lipids In the case of Ab40, we consider this disorder primarily to be the result of two factors: (a) a reduction in the area per lipid headgroup and (b) the unfolding of the C-terminal, embedded, region of the peptide As discussed above, Ab40 is capable of increasing the tilt of the surrounding lipids, thus disordering their acyl chains The fact that melittin also causes a similar amount of disorder on the surrounding lipids leads to an interesting question If, as we have proposed, melittin does not interact as strongly as Ab with the lipid FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS 3071 Membrane perturbation by Alzheimer’s Ab J A Lemkul and D R Bevan headgroups, how can we account for the fact that both of these peptides can disorder the surrounding lipids to the same extent? One possible answer to this question was proposed by the authors of the original melittin simulations in which the peptide was embedded in the membrane [18] They observed that the surrounding lipids tend to pack around the peptide and tilt their acyl chains, as the protein itself tilts Another contribution to the disordering effect is the restriction of motion along the acyl chains once the lipids have packed around the protein Because this behavior arises as a result of the tilt of the peptide and interactions between the embedded region of the peptide and the acyl chains, it presents an interesting insight into the interactions between asymmetrically-embedded peptides and nearby lipids Similar to melittin, Ab40 is embedded asymmetrically in the lipid bilayer and, over time, tilts with respect to the bilayer normal As such, we attribute some of the disorder experienced by the nearby lipids to the motion and tilting of this embedded segment Thinning of the bilayer in the presence of Ab40 Because Ab40 is known to disrupt the integrity of the lipid membrane [11,12], another parameter of interest is the local thickness of the bilayer We previously reported the capacity of water to penetrate into the bilayer when Ab40 is present [25], and a more thorough examination of the bilayer thickness is now appropriate in the context of lipid parameters The experimentally-determined thickness of a fluidphase DPPC bilayer (in terms of the P–P distance) is 3.7 nm [38] We achieve good agreement with this value in all of our control simulations conducted at 323 K (Fig 2) In the presence of Ab40 (at 323 K), however, the bilayer may become depressed between 1.0 and 1.5 nm, as determined by averaging the bilayer dimensions over the last 25 ns of each simulation As with the reduced area per lipid headgroup, the decreased bilayer thickness is independent of the peptide protonation state Local thinning of the bilayer, of comparable magnitude, is observed in all of the Ab40-DPPC simulations Similar thinning of the bilayer also occurs in the presence of melittin Although the bilayer may deform to decrease its thickness up to 1.0 nm (also determined by the same averaging discussed above), the deformed region is much smaller than that in the case of Ab40 We attribute this observation to the fact that, unlike Ab40, melittin retains much of its secondary structure throughout the trajectory, thus remaining more 3072 compact The unfolding of Ab40 makes it more accessible to a wider area, and thus more lipids, which leads to a more pronounced depression in the bilayer Hydrophobic mismatch most likely plays a part in the local deformation of the bilayer Systematic analysis of hydrophobic mismatch using KALP model peptides by Kandasamy and Larson [22] illustrates that short, helical peptides, with charges placed within the lipid headgroups, can result in depression of bilayer thickness or tilting of the peptide to accommodate the size of the peptide The systems reported in the present study also include elements of hydrophobic mismatch because neither Ab40 nor melittin completely span the bilayer in their initial configurations This orientation positions the hydrophobic, embedded, regions of the peptide approximately halfway through the bilayer Thus, it is not unreasonable to conclude that the bilayer deforms to accommodate this orientation Even as the simulations progress, and the peptides alter their orientation, becoming more deeply embedded in some cases, the length of the short hydrophobic stretch remains, and is less than the dimensions of the hydrophobic core of the bilayer This mismatch, anchored at the water–bilayer interface in many cases by charged amino acids such as Lys16 and Lys28 in Ab40, likely contributes to the deformation of the dimensions of the surrounding bilayer The observations made from our simulations compare well with experimental observations An early study by Mason et al [7] indicated that Ab40 was capable of penetrating into rat synaptic plasma membranes, thus decreasing bilayer thickness In addition, Kayed et al [11] report that, although oligomeric Ab species are primarily responsible for membrane permeability, monomeric and low molecular weight species can penetrate into the bilayer interior and cause thinning in the order of 0.5 nm Although the pathogenic agent of Alzheimer’s disease is widely believed to be an oligomeric Ab species, it is also important to understand the interactions of monomeric Ab with lipid bilayers The results reported in the present study potentially shed light on the molecular interactions that give rise to the experimentally-observed behaviors described above, including a new hypothesis for a functional role of the 16 N-terminal residues of Ab Although it has long been postulated that Ab is partially embedded in the hydrophobic core of the bilayer via its C-terminus, a functional role for its N-terminus has not yet been established Our simulations suggest an important role for the N-terminus in associating with the surrounding lipid matrix The preponderance of charged amino acid side chains and the exposure of the disordered backbone interact favorably with the FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS J A Lemkul and D R Bevan Membrane perturbation by Alzheimer’s Ab strongly polar environment of the membrane–water interface It is not surprising that this region of the peptide may contribute to neurotoxicity The N-terminal sequence is cleaved by a-secretase to generate the non-amyloidogenic sAPPa and p3 fragments from amyloid precursor protein, precluding the production of Ab [43,44] The p3 peptide and Ab differ by only the absence or presence of these 16 N-terminal residues, and p3 is nontoxic, whereas Ab is neurotoxic The toxicity of Ab has been proposed to be exerted through its interactions with membranes [45], suggesting that this sequence of amino acids likely plays an important role in Ab–membrane interactions In the present study, we report the molecular basis for the perturbation of lipids, which is a result of both hydrophobic mismatch of the C-terminal region of Ab within the lipid bilayer and the favorable interaction of the N-terminal segment of Ab with the polar environment of the lipid headgroups Understanding this simple system (i.e monomeric Ab in a model membrane) provides an excellent starting point for the study of more complex systems of oligomeric Ab peptides in membrane systems Following NVT equilibration, isothermal–isobaric (NPT) equilibration was performed for 500 ps, applying a pressure of 10 MPa in the transverse direction and 0.1 MPa in the vertical direction The pressure of the system was regulated anisotropically using the Berendsen barostat [46] with a relaxation time (sP) of 2.0 ps These conditions were employed to accelerate packing of the lipids around the peptide (if present) and applied to the pure DPPC systems for consistency The same position restraints and simulation parameters applied in the NVT step were also used during NPT equilibration Experimental procedures Acknowledgements Equilibration The authors thank W J Allen for contributing the analysis scripts used in the present study, the administrators of Advanced Research Computing at Virginia Tech (SystemX) for computing time and technical support, and the anonymous reviewers, whose comments substantially improved the quality of this paper All simulations were performed using the gromacs software package, version 3.3 [28] All systems were equilibrated under an isothermal–isochoric (NVT) ensemble for 100 ps Position restraints were placed on all peptide heavy atoms (if present) in all directions, and on the phosphorus atoms of the lipid headgroups in the vertical direction All position restraints utilized a spring constant, kpr, of 1000 kJỈmol)1Ỉnm)2 The Berendsen thermostat [46] was used to regulate temperature, with a relaxation time (sT) of 0.1 ps Each group (protein, lipids, solvent ⁄ ions) was coupled to a separate temperature bath The parameters developed by Berger et al [47] were applied to the DPPC lipids, and the gromos96 53a6 parameter set was used to describe the rest of the system (protein, solvent, ions) Lennard– Jones interactions were cut-off at 1.4 nm, and short-range, nonbonded interactions were calculated with a twin-range cut-off scheme (0.9 ⁄ 1.4 nm), with the neighbor list updated every five simulation steps Long-range electrostatic interactions were calculated using the particle mesh Ewald method [48] with fourth-order spline interpolation and a Fourier grid spacing of 0.12 nm This treatment of electrostatics has been shown to provide an accurate representation of lipid properties [49], and is also commonly used in simulations of proteins The linear constraint solver method [50] was used to constrain all bond lengths, allowing a fs integration step Production MD Following the 600 ps of equilibration, production MD was conducted for 100 ns, using an NPT ensemble A pressure of 0.1 MPa was applied in all directions with all other parameters being the same as in the NPT equilibration All position restraints were removed prior to the production phase Simulations were conducted using Virginia Tech’s SystemX supercomputer (2.3 GHz PowerPC 970FX processors) Coordinates were saved every ps for analysis All analyses were performed using tools within the gromacs software package or code developed in-house References Alzheimer A (1991) Arteriosclerotic brain atrophy Int J Geriatr Psychiatry 6, 129–130 Haass C & Selkoe DJ (2007) Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid b-peptide Nat Rev Mol Cell Biol 8, 101–112 Saido TC & Iwata N (2006) Metabolism of amyloid b peptide and pathogenesis of Alzheimer’s disease: towards presymptomatic diagnosis, prevention and therapy Neurosci Res 54, 235–253 Coles M, Bicknell W, Watson AA, Fairlie DP & Craik DJ (1998) Solution structure of amyloid b-peptide(1-40) in a water-micelle environment Is the membrane-spanning domain where we think it is? Biochemistry 37, 11064–11077 Tischer E & Cordell B (1996) b-amyloid precursor protein: location of transmembrane domain and specificity of c-secretase cleavage J Biol Chem 271, 21914–21919 FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS 3073 Membrane perturbation by Alzheimer’s Ab J A Lemkul and D R Bevan Yip CM & McLaurin J (2001) Amyloid-b peptide assembly: a critical step in fibrillogenesis and membrane disruption Biophys J 80, 1359–1371 Mason RP, Jacob RF, Walter MF, Mason PE, Avdulov NA, Chochina SV, Igbavboa U & Wood WG (1999) Distribution and fluidizing action of soluble and aggregated amyloid b-peptide in rat synaptic plasma membranes J Biol Chem 274, 18801–18807 Avdulov NA, Chochina SV, Igbavboa U, Warden CS, Vassiliev AV & Wood WG (1997) Lipid binding to amyloid b-peptide aggregates: preferential binding of cholesterol as compared with phosphatidylcholine and fatty acids J Neurochem 69, 1746–1752 Muller WE, Kirsch C & Eckert GP (2001) Membraneă disordering effects of b-amyloid peptides Biochem Soc Trans 29, 617–623 10 Mattson MP, Cheng B, Davis D, Bryant K, Lieberburg I & Rydel RE (1992) b-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity J Neurosci 12, 376–389 11 Kayed R, Sokolov Y, Edmonds B, McIntire TM, Milton SC, Hall JE & Glabe CG (2004) Permeabilization of lipid bilayers is a common conformation-dependent activity of soluble amyloid oligomers in protein misfolding diseases J Biol Chem 279, 46363–46366 12 Ambroggio EE, Kim DH, Separovic F, Barrow CJ, Barnham KJ, Bagatolli LA & Fidelio GD (2005) Surface behavior and lipid interaction of Alzheimer b-amyloid peptide 1-42: a membrane-disrupting peptide Biophys J 68, 2706–2713 13 Terwilliger TC & Eisenberg D (1982) The structure of melittin II Interpretation of the structure J Biol Chem 257, 6016–6022 14 Vogel H & Jahnig F (1986) The structure of melittin in ă membranes Biophys J 50, 573–582 15 Dempsey CE (1990) The actions of melittin on membranes Biochim Biophys Acta 1031, 143–161 ` 16 Berneche S, Nina M & Roux B (1998) Molecular dynamics simulation of melittin in a dimyristoylphosphatidylcholine bilayer membrane Biophys J 75, 1603–1618 17 Bachar M & Becker OM (1999) Melittin at a membrane ⁄ water interface: effects on water orientation and water penetration J Chem Phys 111, 8672–8685 18 Bachar M & Becker OM (2000) Protein-induced membrane disorder: a molecular dynamics study of melittin in a dipalmitoylphosphatidylcholine bilayer Biophys J 78, 1359–1375 19 Bond PJ & Sansom MSP (2007) Bilayer deformation by the Kv channel voltage sensor domain revealed by selfassembly simulations Proc Natl Acad Sci USA 104, 2631–2636 20 Khalid S & Sansom MSP (2006) Molecular dynamics simulations of a bacterial autotransporter: NalP from Neisseria meningitidis Mol Membr Biol 23, 499–508 3074 21 Deol SS, Bond PJ, Domene C & Sansom MSP (2004) Lipid-protein interactions of integral membrane proteins: a comparative simulation study Biophys J 87, 3737–3749 22 Kandasamy SK & Larson RG (2006) Molecular dynamics simulations of model Trans-membrane peptides in lipid bilayers: a systematic investigation of hydrophobic mismatch Biophys J 90, 2326–2343 23 Sperotto MM, May S & Baumgaertner A (2006) Modelling of proteins in membranes Chem Phys Lipids 141, 2–29 24 Xu Y, Shen J, Luo X, Zhu W, Chen K, Ma J & Jiang H (2005) Conformational transition of the amyloid b-peptide Proc Natl Acad Sci USA 102, 5403–5407 25 Lemkul JA & Bevan DR (2008) A comparative molecular dynamics analysis of the amyloid b-peptide in a lipid bilayer Arch Biochem Biophys 470, 54–63 26 Davis CH & Berkowitz ML (2009) Interaction between amyloid-b (1-42) peptide and phospholipid bilayers: a molecular dynamics study Biophys J 96, 785–797 27 Tieleman DP & Berendsen HJC (1996) Molecular dynamics simulations of a fully hydrated dipalmitoylphosphatidylcholine bilayer with different macroscopic boundary conditions and parameters J Chem Phys 105, 4871–4880 28 van der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE & Berendsen HJC (2005) GROMACS: fast, flexible, and free J Comput Chem 26, 1701–1718 29 Allen WJ, Lemkul JA & Bevan DR (2009) GridMATMD: a grid-based membrane analysis tool for use with molecular dynamics J Comput Chem doi: 10.1002/ jcc.21172 (in press) 30 Terwilliger TC & Eisenberg D (1982) The structure of melittin I Structure determination and partial refinement J Biol Chem 257, 6010–6015 31 Bradrick TD, Philippetis A & Georghiou S (1995) Stopped-flow fluorometric study of the interaction of melittin with phospholipid bilayers: importance of the physical state of the bilayer and the acyl chain length Biophys J 69, 1999–2010 32 Bradshaw JP, Dempsey CE & Watts A (1994) A combined x-ray and neutron diffraction study of selectively deuterated melittin in phospholipid bilayers: effect of pH Mol Membr Biol 11, 79–86 33 Monette M & Lafleur M (1995) Modulation of melittininduced lysis by surface charge density of membranes Biophys J 68, 187–195 34 Ohki S, Marcus E, Sukumaran DK & Arnold K (1994) Interaction of melittin with lipid membranes Biochim Biophys Acta 1112, 1–6 35 Tieleman DP, Forrest LR, Sansom MSP & Berendsen HJC (1998) Lipid properties and the orientation of aromatic residues in OmpF, influenza M2, and alamethicin systems: molecular dynamics simulations Biochemistry 37, 17554–17561 FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS J A Lemkul and D R Bevan ´ 36 Douliez J-P, Leonard A & Dufourc EJ (1995) Restatement of order parameters in biomembranes: calculation of C-C bond order parameters from C-D quadrupolar splittings Biophys J 68, 1727–1739 37 Tieleman DP, Marrink SJ & Berendsen HJC (1997) A computer perspective of membranes: molecular dynamics studies of lipid bilayer systems Biochim Biophys Acta 1331, 235–270 38 Lewis BA & Engelman DM (1983) Lipid bilayer thickness varies linearly with acyl chain length in fluid phosphatidylcholine vesicles J Mol Biol 166, 211–217 39 Nagle JF (1993) Area ⁄ lipid of bilayers from NMR Biophys J 64, 1476–1481 40 Nagle JF & Tristam-Nagle S (2000) Structure of lipid bilayers Biochim Biophys Acta 1469, 159–195 41 Pandit SA, Bostick D & Berkowitz ML (2003) Molecular dynamics simulation of a dipalmitoylphosphatidylcholine bilayer with NaCl Biophys J 84, 3743–3750 42 Petrache HI, Tu K & Nagle JF (1999) Analysis of simulated NMR order parameters for lipid bilayer structure determination Biophys J 76, 2479–2487 43 Selkoe DJ (1999) Translating cell biology into therapeutic advances in Alzheimer’s disease Nature 399, A23–A31 44 Thinakaran G & Koo EH (2008) Amyloid precursor protein trafficking, processing, and function J Biol Chem 283, 29615–29619 45 Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW & Glabe CG (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis Science 300, 486–489 46 Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A & Haak JR (1984) Molecular dynamics with coupling to an external bath J Chem Phys 81, 3684– 3690 47 Berger O, Edholm O & Jahnig F (1997) Molecular ă dynamics simulations of a uid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature Biophys J 72, 2002– 2013 48 Essmann U, Perera L, Berkowitz ML, Darden T, Lee H & Pedersen LG (1995) A smooth particle mesh Ewald Method J Chem Phys 103, 8577–8593 49 Patra M, Karttunen M, Hyvonen MT, Falck E, ă Lindqvist P & Vattulainen I (2003) Molecular dynamics simulations of lipid bilayers: major artifacts due to Membrane perturbation by Alzheimer’s Ab truncating electrostatic interactions Biophys J 84, 3636–3645 50 Hess B, Bekker H, Berendsen HJC & Fraaije JGEM (1997) LINCS: a linear constraint solver for molecular simulations J Comput Chem 18, 1463–1472 Supporting information The following supplementary material is available: Fig S1 Density plots Fig S2 Simulation set A Fig S3 Simulation set B, with images rendered as described in Fig S1 Fig S4 Simulation set C, with images rendered as described in Fig S2 Fig S5 Simulation sets E and P, with images of the melittin peptide, rendered as described in Fig S2 Fig S6 Simulation sets O and N, showing the progression of bilayer thickness for pure DPPC membrane systems Fig S7 Area per lipid headgroup as a function of time for simulation set B Fig S8 Area per lipid headgroup as a function of time for simulation set C Fig S9 Area per lipid headgroup as a function of time for simulation sets E and P Fig S10 Area per lipid headgroup as a function of distance from the protein: simulations A3 and A4 Fig S11 Area per lipid headgroup as a function of distance from the protein: simulations B1 and B2 Fig S12 Area per lipid headgroup as a function of distance from the protein: simulations B3 and B4 Fig S13 Area per lipid headgroup as a function of distance from the protein: simulations C1 and C2 Fig S14 Area per lipid headgroup as a function of distance from the protein: simulations E1 and E2 Fig S15 Area per lipid headgroup as a function of distance from the protein: simulations P1 and P2 This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 276 (2009) 3060–3075 ª 2009 The Authors Journal compilation ª 2009 FEBS 3075 ... Ma J & Jiang H (2005) Conformational transition of the amyloid b-peptide Proc Natl Acad Sci USA 102, 540 3–5 407 25 Lemkul JA & Bevan DR (2008) A comparative molecular dynamics analysis of the amyloid. .. using a window of ten data points designation In the case of simulation A1 , the area per lipid headgroup is largely constant at the outset of the ˚ simulation, fluctuating around a value of 62 A2 ... represent an average of the plateau region for each leaflet of the bilayer From these data, it can be seen that, overall, the )SCD values in the top leaflet of the Ab40-DPPC systems are lower than those