Embedding Aβ42 in Heterogeneous Membranes Depends on Cholesterol Asymmetries

Using a coarse-grained lipid and peptide model, we show that the free energy stabilization of amyloid-β in heterogeneous lipid membranes is predicted to have a dependence on asymmetric distributions of cholesterol compositions across the membrane leaflets. We find that a highly asymmetric cholesterol distribution that is depleted on the exofacial leaflet but enhanced on the cytofacial leaflet of the model lipid membrane thermodynamically favors membrane retention of a fully embedded Aβ peptide. However, in the case of cholesterol redistribution that increases concentration of cholesterol on the exofacial layer, typical of aging or Alzheimer’s disease, the free energy favors peptide extrusion of the highly reactive N-terminus into the extracellular space that may be vulnerable to aggregation, oligomerization, or deleterious oxidative reactivity.     

Differences in β-strand Populations of Monomeric Aβ40 and Aβ42

Using homonuclear ¹H NOESY spectra, with chemical shifts, ³J_H^N_H^α scalar couplings, residual dipolar couplings, and ¹H-¹⁵N NOEs, we have optimized and validated the conformational ensembles of the amyloid-β 1–40 (Aβ40) and amyloid-β 1–42 (Aβ42) peptides generated by molecular dynamics simulations. We find that both peptides have a diverse set of secondary structure elements including turns, helices, and antiparallel and parallel β-strands. The most significant difference in the structural ensembles of the two peptides is the type of β-hairpins and β-strands they populate. We find that Aβ42 forms a major antiparallel β-hairpin involving the central hydrophobic cluster residues (16–21) with residues 29–36, compatible with known amyloid fibril forming regions, whereas Aβ40 forms an alternative but less populated antiparallel β-hairpin between the central hydrophobic cluster and residues 9–13, that sometimes forms a β-sheet by association with residues 35–37. Furthermore, we show that the two additional C-terminal residues of Aβ42, in particular Ile-41, directly control the differences in the β-strand content found between the Aβ40 and Aβ42 structural ensembles. Integrating the experimental and theoretical evidence accumulated over the last decade, it is now possible to present monomeric structural ensembles of Aβ40 and Aβ42 consistent with available information that produce a plausible molecular basis for why Aβ42 exhibits greater fibrillization rates than Aβ40.     

Retardation of Aβ Fibril Formation by Phospholipid Vesicles Depends on Membrane Phase Behavior

An increasing amount of evidence suggests that in several amyloid diseases, the fibril formation in vivo and the mechanism of toxicity both involve membrane interactions. We have studied Alzheimer's disease related amyloid beta peptide (Aβ). Recombinant Aβ(M1-40) and Aβ(M1-42) produced in Escherichia coli, allows us to carry out large scale kinetics assays with good statistics. The amyloid formation process is followed in means of thioflavin T fluorescence at relatively low (down to 380 nM) peptide concentration approaching the physiological range. The lipid membranes are introduced in the system as large and small unilamellar vesicles. The aggregation lagtime increases in the presence of lipid vesicles for all situations investigated and the phase behavior of the membrane in the vesicles has a large effect on the aggregation kinetics. By comparing vesicles with different membrane phase behavior we see that the solid gel phase dipalmitoylphosphatidylcholine bilayers cause the largest retardation of Aβ fibril formation. The membrane-induced retardation reaches saturation and is present when the vesicles are added during the lag time up to the nucleation point. No significant difference is detected in lag time when increasing amount of negative charge is incorporated into the membrane.     

Effects of β-Cyclodextrin on the Structure of Sphingomyelin/Cholesterol Model Membranes

The interaction of β-cyclodextrin (β-CD) with mixed bilayers composed of sphingomylein and cholesterol (Chol) above and below the accepted stable complexation ratio (67:33) was investigated. Membranes with the same (symmetric) and different (asymmetric) compositions in their inner and outer leaflets were deposited at surface pressures of 20, 30, and 40 mN/m at the solid-liquid interface. Using neutron reflectometry, membranes of various global molar ratios (defined as the sum of the molar ratios of the inner and outer leaflets), were characterized before and after β-CD was added to the subphase. The structure of bilayers with global molar ratios at or above the stable complexation ratio was unchanged by β-CD, indicating that β-CD is unable to remove sphingomyelin or complexed Chol. However, β-CD removed all uncomplexed Chol from bilayers composed of global molar ratios below the stable complexation ratio. The removal of Chol by β-CD was independent of the initial structure of the membranes as deposited, suggesting that asymmetric membranes homogenize by the exchange of molecules between leaflets. The interaction of β-CD with the aforementioned membranes was independent of the deposition surface pressure except for a symmetric 50:50 membrane deposited at 40 mN/m. The scattering from 50:50 bilayers with higher packing densities (deposited at 40 mN/m) was unaffected by β-CD, suggesting that the removal of Chol can depend on both the composition and packing density of the membrane.     

Mapping Conformational Ensembles of Aβ Oligomers in Molecular Dynamics Simulations

Although the oligomers formed by Aβ peptides appear to be the primary cytotoxic species in Alzheimer's disease, detailed information about their structures appears to be lacking. In this article, we use exhaustive replica exchange molecular dynamics and an implicit solvent united-atom model to study the structural properties of Aβ monomers, dimers, and tetramers. Our analysis suggests that the conformational ensembles of Aβ dimers and tetramers are very similar, but sharply distinct from those sampled by the monomers. The key conformational difference between monomers and oligomers is the formation of β-structure in the oligomers occurring together with the loss of intrapeptide interactions and helix structure. Our simulations indicate that, independent of oligomer order, the Aβ aggregation interface is largely confined to the sequence region 10-23, which forms the bulk of interpeptide interactions. We show that the fractions of β structure computed in our simulations and measured experimentally are in good agreement.     

New lignans attenuating cognitive deterioration of Aβ transgenic flies discovered in Acorus tatarinowii

Five new lignan racemates, (±)-tatarinoid D–H (1–5), and three known analogues (6–8) were isolated from the rhizomes of Acorus tatarinowii. Their structures were established by 1D, 2D-NMR, HR-MS, IR and optical spectral data. Among them, 1 and 6–8 can alleviate the cognitive deterioration of Aβ transgenic flies.     

Replica Exchange Simulations of the Thermodynamics of Aβ Fibril Growth

Replica exchange molecular dynamics and an all-atom implicit solvent model are used to probe the thermodynamics of deposition of Alzheimer's Aβ monomers on preformed amyloid fibrils. Consistent with the experiments, two deposition stages have been identified. The docking stage occurs over a wide temperature range, starting with the formation of the first peptide-fibril interactions at 500 K. Docking is completed when a peptide fully adsorbs on the fibril edge at the temperature of 380 K. The docking transition appears to be continuous, and occurs without free energy barriers or intermediates. During docking, incoming Aβ monomer adopts a disordered structure on the fibril edge. The locking stage occurs at the temperature of ≈360 K and is characterized by the rugged free energy landscape. Locking takes place when incoming Aβ peptide forms a parallel β-sheet structure on the fibril edge. Because the β-sheets formed by locked Aβ peptides are typically off-registry, the structure of the locked phase differs from the structure of the fibril interior. The study also reports that binding affinities of two distinct fibril edges with respect to incoming Aβ peptides are different. The peptides bound to the concave edge have significantly lower free energy compared to those bound on the convex edge. Comparison with the available experimental data is discussed.     

Molecular Dynamics Simulations of Ibuprofen Binding to Aβ Peptides

Using replica exchange molecular dynamics simulations and the implicit solvent model we probed binding of ibuprofen to Aβ_10-40 monomers and amyloid fibrils. We found that the concave (CV) fibril edge has significantly higher binding affinity for ibuprofen than the convex edge. Furthermore, binding of ibuprofen to Aβ monomers, as compared to fibrils, results in a smaller free energy gain. The difference in binding free energies is likely to be related to the presence of the groove on the CV fibril edge, in which ibuprofen tends to accumulate. The confinement effect of the groove promotes the formation of large low-energy ibuprofen clusters, which rarely occur on the surface of Aβ monomers. These observations led us to suggest that the ibuprofen binding mechanism for Aβ fibrils is different from that for monomers. In general, ibuprofen shows a preference to bind to those regions of Aβ monomers (amino terminal) and fibrils (the CV edge) that are also the primary aggregation interfaces. Based on our findings and on available experimental data, we propose a rationale for the ibuprofen antiaggregation effect.     

Probing Energetics of Aβ Fibril Elongation by Molecular Dynamics Simulations

Using replica exchange molecular dynamics simulations and an all-atom implicit solvent model, we probed the energetics of Aβ_10-40 fibril growth. The analysis of the interactions between incoming Aβ peptides and the fibril led us to two conclusions. First, considerable variations in fibril binding propensities are observed along the Aβ sequence. The peptides in the fibril and those binding to its edge interact primarily through their N-termini. Therefore, the mutations affecting the Aβ positions 10-23 are expected to have the largest impact on fibril elongation compared with those occurring in the C-terminus and turn. Second, we performed weak perturbations of the binding free energy landscape by scanning partial deletions of side-chain interactions at various Aβ sequence positions. The results imply that strong side-chain interactions—in particular, hydrophobic contacts—impede fibril growth by favoring disordered docking of incoming peptides. Therefore, fibril elongation may be promoted by moderate reduction of Aβ hydrophobicity. The comparison with available experimental data is presented.     

Two Distinct Conformations of Aβ Aggregates on the Surface of Living PC12 Cells

Aβ42 has been found to associate rapidly to neuronal cells and is the primary constituent of senile plaques. In this study we monitored the aggregation of Aβ42 with living PC12 cells. Using photobleaching Förster resonance energy transfer, we observed one set of aggregates that displayed colocalization and another that displayed energy transfer. Cell surface aggregates were found to become resistant to potassium iodide (KI)-induced quenching. Exposed Aβ42 regions were probed with three monoclonal antibodies directed against the N-terminus, an internal sequence, and the C-terminus of Aβ42. Two populations of aggregates were revealed: one that bound all three antibodies, and one that bound all but the C-terminus antibody. Of interest, using fluorescent recovery after photobleaching, we observed no Aβ42 exchange within either type of aggregate. These findings offer what we believe is new insight into the conformations of Aβ42 that accumulate on the surface of living cells. One conformation is incapable of energy transfer, is sensitive to KI, and binds C-terminus-specific antibodies. The other conformation increases in number over time, is capable of energy transfer, is quencher-resistant, and has a sequestered C-terminus. With further studies to characterize Aβ aggregation on live cells, the underlying mechanisms leading to Alzheimer's disease may be revealed.     

Aβ Oligomers and Fibrillar Aggregates Induce Different Apoptotic Pathways in LAN5 Neuroblastoma Cell Cultures

Fibril deposit formation of amyloid β-protein (Aβ) in the brain is a hallmark of Alzheimer's disease (AD). Increasing evidence suggests that toxicity is linked to diffusible Aβ oligomers, which have been found in soluble brain extracts of AD patients, rather than to insoluble fibers. Here we report a study of the toxicity of two distinct forms of recombinant Aβ small oligomers and fibrillar aggregates to simulate the action of diffusible Aβ oligomers and amyloid plaques on neuronal cells. Different techniques, including dynamic light scattering, fluorescence, and scanning electron microscopy, have been used to characterize the two forms of Aβ. Under similar conditions and comparable incubation times in neuroblastoma LAN5 cell cultures, oligomeric species obtained from Aβ peptide are more toxic than fibrillar aggregates. Both oligomers and aggregates are able to induce neurodegeneration by apoptosis activation, as demonstrated by TUNEL assay and Hoechst staining assays. Moreover, we show that aggregates induce apoptosis by caspase 8 activation (extrinsic pathway), whereas oligomers induce apoptosis principally by caspase 9 activation (intrinsic pathway). These results are confirmed by cytochrome c release, almost exclusively detected in the cytosolic fraction of LAN5 cells treated with oligomers. These findings indicate an active and direct interaction between oligomers and the cellular membrane, and are consistent with internalization of the oligomeric species into the cytosol.     

Alzheimer's Aβ(1-40) Amyloid Fibrils Feature Size-Dependent Mechanical Properties

Amyloid fibrils are highly ordered protein aggregates that are associated with several pathological processes, including prion propagation and Alzheimer''s disease. A key issue in amyloid science is the need to understand the mechanical properties of amyloid fibrils and fibers to quantify biomechanical interactions with surrounding tissues, and to identify mechanobiological mechanisms associated with changes of material properties as amyloid fibrils grow from nanoscale to microscale structures. Here we report a series of computational studies in which atomistic simulation, elastic network modeling, and finite element simulation are utilized to elucidate the mechanical properties of Alzheimer''s Aβ(1-40) amyloid fibrils as a function of the length of the protein filament for both twofold and threefold symmetric amyloid fibrils. We calculate the elastic constants associated with torsional, bending, and tensile deformation as a function of the size of the amyloid fibril, covering fibril lengths ranging from nanometers to micrometers. The resulting Young''s moduli are found to be consistent with available experimental measurements obtained from long amyloid fibrils, and predicted to be in the range of 20–31 GPa. Our results show that Aβ(1-40) amyloid fibrils feature a remarkable structural stability and mechanical rigidity for fibrils longer than ≈100 nm. However, local instabilities that emerge at the ends of short fibrils (on the order of tens of nanometers) reduce their stability and contribute to their disassociation under extreme mechanical or chemical conditions, suggesting that longer amyloid fibrils are more stable. Moreover, we find that amyloids with lengths shorter than the periodicity of their helical pitch, typically between 90 and 130 nm, feature significant size effects of their bending stiffness due the anisotropy in the fibril''s cross section. At even smaller lengths (⪅50 nm), shear effects dominate lateral deformation of amyloid fibrils, suggesting that simple Euler-Bernoulli beam models fail to describe the mechanics of amyloid fibrils appropriately. Our studies reveal the importance of size effects in elucidating the mechanical properties of amyloid fibrils. This issue is of great importance for comparing experimental and simulation results, and gaining a general understanding of the biological mechanisms underlying the growth of ectopic amyloid materials.     

Human Serum Albumin Inhibits Aβ Fibrillization through a “Monomer-Competitor” Mechanism

Human serum albumin (HSA) is not only a fatty acid and drug carrier protein, it is also a potent inhibitor of Aβ self-association in plasma. However, the mechanism underlying the inhibition of Aβ fibrillization by HSA is still not fully understood. We therefore investigated the Aβ-HSA system using a combined experimental strategy based on saturation transfer difference (STD) NMR and intrinsic albumin fluorescence experiments on three Aβ peptides with different aggregation propensities (i.e., Aβ(12-28), Aβ(1-40), and Aβ(1-42)). Our data consistently show that albumin selectively binds to cross-β-structured Aβ oligomers as opposed to Aβ monomers. The HSA/Aβ oligomer complexes have K_D values in the micromolar to submicromolar range and compete with the further addition of Aβ monomers to the Aβ assemblies, thus inhibiting fibril growth (“monomer competitor” model). Other putative mechanisms, according to which albumin acts as a “monomer stabilizer” or a “dissociation catalyst”, are not supported by our data, thus resolving previous discrepancies in the literature regarding Aβ-HSA interactions. In addition, the model and the experimental approaches proposed here are anticipated to have broad relevance for the characterization of other systems that involve amyloidogenic peptides and oligomerization inhibitors.     

Induced β-Barrel Formation of the Alzheimer's Aβ25-35 Oligomers on Carbon Nanotube Surfaces: Implication for Amyloid Fibril Inhibition

Recent experimental studies show that carbon nanotubes impact the aggregation process of proteins associated with neurodegenerative diseases. However, the details of molecular interactions between proteins and carbon nanotubes are still not well understood. In this study, we investigate the initial adsorption features and dynamics of the Alzheimer's amyloid-β peptide spanning residues 25-35 (Aβ25-35) on a single-walled carbon nanotube (SWNT) surface using fully atomic molecular dynamics simulations (MD) in explicit solvent. The initial configurations of the Aβ25-35 peptides consist of two preformed bilayer β-sheets, each with four or five β-strands in parallel or mixed antiparallel-parallel orientations. Our simulations show, for what we believe is the first time, that two disjointed Aβ25-35 β-sheets with mixed antiparallel-parallel strands can assemble into β-barrels wrapping the SWNT. In contrast, both simulations of Aβ25-35 without SWNT, and simulations of SWNT−Aβ25-35 with purely parallel β-strands, lead to disordered aggregates. We find that Aβ25-35 β-barrel formation involves at least two steps: i), curving of the Aβ25-35 β-sheets as a result of strong hydrophobic interactions with carbon nanotube concomitantly with dehydration of the SWNT-peptide interface; and ii), intersheet backbone hydrogen bond formation with fluctuating intrasheet hydrogen bonds. Detailed analysis of the conversion shows that β-barrel formation on SWNT surface results from the interplay of dehydration and peptide-SWNT/peptide-peptide interactions. Implications of our results on amyloid fibril inhibition are discussed.     

Synergistic Insertion of Antimicrobial Magainin-Family Peptides in?Membranes Depends on the Lipid Spontaneous Curvature

PGLa and magainin 2 (MAG2) are amphiphilic antimicrobial peptides from frog skin with known synergistic activity. The orientation of the two helices in membranes was studied using solid-state ¹⁵N-NMR, for each peptide alone and for a 1:1 mixture of the peptides, in a range of different lipid systems. Two types of orientational behavior emerged. 1), In lipids with negative spontaneous curvature, both peptides remain flat on the membrane surface, when assessed both alone and in a 1:1 mixture. 2), In lipids with positive spontaneous curvature, PGLa alone assumes a tilted orientation but inserts into the bilayer in a transmembrane alignment in the presence of MAG2, whereas MAG2 stays on the surface or gets only slightly tilted, when observed both alone and in the presence of PGLa. The behavior of PGLa alone is identical to that of another antimicrobial peptide, MSI-103, in the same lipid systems, indicating that the curvature-dependent helix orientation is a general feature of membrane-bound peptides and also influences their synergistic intermolecular interactions.The two antimicrobial peptides PGLa and magainin 2 (MAG2) from the African frog Xenopus laevis, which are active against Gram-positive and Gram-negative bacteria, show intriguing synergistic effects that are not yet well understood (1). Structural insights into this synergy may help in the development of a new antibiotic-combination therapy. Both peptides are known to form α-helices when bound to lipid bilayers (2–4). The orientation of such α-helices in a membrane can be readily determined from the ¹⁵N-NMR chemical shift in oriented lipid bilayers that are aligned with the sample normal parallel to the external magnetic field (5). If the ¹⁵N chemical shift is ∼90 ppm, the peptide lies flat on the membrane surface (in the so-called S-state). On the other hand, when the ¹⁵N chemical shift is ∼200 ppm, the peptide is fully inserted (the I-state) in a transmembrane alignment. For intermediate orientations, where the peptide is tilted (the T-state) with an angle of typically 30°–60° relative to the membrane normal, intermediate chemical shifts are expected, but the exact tilt angle cannot be determined from a single label in such cases (6). Using several selectively ²H- or ¹⁹F-labeled peptide analogs, more exact orientations can be obtained, since both the tilt and the azimuthal angles can be measured with high accuracy, and valuable information about dynamics also can be deduced (3,7–10).The orientation of PGLa and MAG2 in membranes has been extensively studied with solid-state NMR, and some clues about the synergistic mechanism have been observed. Notably, in DMPC/DMPG membranes, it has been shown that the peptides on their own are in the S-state or T-state, but when the peptides are mixed in a 1:1 molar ratio, PGLa changes to the I-state, whereas MAG2 stays on the membrane surface (1,11,12). Thus, in the mixed system, transmembrane pores, which would not be spontaneously formed by each peptide on its own, appear to be stable, which could be the basis for synergy. On the other hand, it was also reported that in POPC/POPG there is no change in orientation when PGLa and MAG2 are mixed, as both peptides stay always in the S-state (12). This observation was attributed to the greater hydrophobic thickness of the POPC/POPG bilayer, compared to the DMPC/DMPG bilayer, suggesting that the PGLa helix is so short that it can only insert into thin DMPC/DMPG membranes. However, we have recently shown for MSI-103, a designer-made antimicrobial peptide based on the PGLa sequence, that the orientation determined by ²H-NMR depends not on the bilayer thickness but rather on the intrinsic spontaneous curvature of the lipids (13). Accordingly, an insertion of PGLa and MAG2 into POPC/POPG should be prevented by the pronounced negative spontaneous curvature induced by the unsaturated acyl chains. However, a simple comparison of only two lipid systems does not yield an answer as to which of these two hypotheses is correct. Therefore, we have now collected data over a wide range of lipid systems, with systematic variations of acyl chain lengths (to address bilayer thickness) as well as chain saturations (to address lipid curvature) (see Table S1 in the Supporting Material). In this way, we found unambiguously that lipid curvature is also the decisive factor in the insertion of PGLa/MAG2.Here, ¹⁵N-NMR spectra of singly labeled peptides were recorded, and for each liquid-crystalline lipid system we prepared four oriented samples: ¹⁵N-MAG2 alone, ¹⁵N-MAG2 with PGLa, ¹⁵N-PGLa with MAG2, and ¹⁵N-PGLa alone. The total peptide/lipid molar ratio (P/L) was 1:50. ¹⁵N-NMR spectra are shown in Fig. 1, and the chemical shifts are listed in Fig. 2. The quality of each oriented sample was checked with ³¹P-NMR (see Fig. S1). Our previous detailed ²H- and ¹⁹F-NMR analysis of PGLa in DMPC and DMPC/DMPG showed that the helix realigns depending on the peptide concentration. Namely, at low concentration, PGLa is in an S-state with a tilt angle of ∼98° (3), but above a threshold concentration around P/L = 1:100, it flips into a tilted T-state with a tilt angle of ∼125° (9,14). In the presence of MAG2, it was found that PGLa inserts almost upright in an I-state with a tilt angle of ∼158° (11). The present ¹⁵N-NMR study confirms that in DMPC/DMPG (3:1), PGLa is in the I-state when mixed with MAG2, as indicated by the ¹⁵N chemical shift of 205 ppm. PGLa alone at P/L = 1:50 has a chemical shift of 116 ppm, which corresponds to a tilted orientation, as expected. MAG2 alone is found to be in the S-state (91 ppm), but when it is mixed with PGLa its signal moves to 105 ppm, indicating a small change in the alignment. MAG2 is, however, clearly not inserted like PGLa.Open in a separate windowFigure 1¹⁵N-NMR spectra of ¹⁵N-labeled PGLa or MAG2, alone or in a synergistic 1:1 mixture with the other peptide, in differently oriented lipids. Powder spectra are shown in the top row. Red, green, and blue lines indicate chemical shifts associated with the S-, T-, and I-state orientations, respectively.Open in a separate windowFigure 2Schematic overview of the orientation of PGLa (red), MAG2 (green), and MSI-103 (orange (13)) in different lipids. The corresponding ¹⁵N-NMR chemical shifts (in ppm) of the spectra in Fig. 1 are indicated beneath each peptide.In thin DLPC bilayers (12 carbon atoms in the chains) we see a behavior similar to that in DMPC (14 carbons), even though the exact chemical shifts are slightly different. PGLa alone is in the T-state, but in combination with MAG2, it flips into the I-state. MAG2, on the other hand, stays in the S-state with and without PGLa. In DPPC bilayers (16 carbons) also, the behavior is similar. PGLa alone is in the T-state but flips into the I-state in the presence of MAG2. MAG2 is slightly more tilted than in DMPC, but it never reaches the I-state.In contrast, in unsaturated lipids, both peptides are always in the S-state, both alone and in the presence of the synergistic partner. In POPC/POPG (9:1), the ¹⁵N chemical shifts of both PGLa and MAG2, alone and in the 1:1 mixture, are between 84 and 89 ppm, clearly indicating a flat alignment on the bilayer surface. As there are no changes in chemical shift with or without the other peptide, this could indicate that there are no interactions between them, in contrast to the situation in saturated lipids. Also, in thin DMoPC bilayers (with 14 carbon atoms and a double bond), the chemical shifts of all samples show that both peptides remain always in the S-state, whether alone or mixed.These results clearly demonstrate that the hydrophobic membrane thickness is not a critical factor for the insertion of PGLa in the presence of MAG2. In DMoPC (thinner than DMPC), there is no insertion, whereas in DPPC (thicker than POPC) insertion occurs. On the other hand, the results fully support the lipid-curvature hypothesis, which states that peptides remain on the surface in membranes composed of lipids with a negative spontaneous curvature, but are more easily tilted or inserted when the lipids have a positive spontaneous curvature (13).In a special lipid mixture, POPE/POPG/TOCL (72:23:5), often used to mimic the composition of the inner membrane of Escherichia coli (15), the result is practically the same as in POPC/POPG (9:1). Also here, chemical shifts of ∼84 ppm indicate that PGLa and MAG2 are always in the S-state, both alone and as a mixture. This behavior is in accordance with the curvature hypothesis, since PE and CL both have a strong negative curvature. On the other hand, when lyso-MPC is added to DMPC to increase the positive curvature, the chemical shift of MAG2 increases to 117 ppm, indicating a more tilted orientation in the membrane with enhanced curvature compared to DMPC or DMPC/DMPG, both with and without PGLa. PGLa alone gives a somewhat larger chemical shift but stays in the T-state, whereas PGLa together with MAG2 flips again into the I-state.We can now compare the results presented here with those from our previous study on the related peptide MSI-103 (13) to find strong correlations. Fig. 2 gives an overview of all results, illustrating the peptide orientations in the different lipid systems. PGLa on its own behaves just like MSI-103 and assumes the same S-state or T-state in the same systems, in full accordance with the lipid-curvature hypothesis. MAG2 alone behaves similarly but seems to have a higher concentration threshold to flip from the S-state to the T-state. In DMPC and DMPC/DMPG, where PGLa is already in the T-state, MAG2 is still in the S-state at P/L = 1:50. However, at P/L = 1:10 (Fig. S2), MAG2 has also reached the T-state. Since MAG2 is charged at both termini, whereas PGLa and MSI-103 are amidated and thus uncharged on the C terminus, it is indeed expected that MAG2 should not start to tilt as easily as PGLa or MSI-103. The polar sector of MAG2 is also larger (Fig. S3).When PGLa and MAG2 are mixed 1:1, their behavior correlates well with that of the individual peptides. In systems where PGLa and MSI-103 are in the S-state, the mixture of PGLa and MAG2 also remains in the S-state. Only when PGLa alone prefers the T-state does it get fully pushed into the I-state by the presence of MAG2. Thus, the model of MAG2-assisted insertion of PGLa proposed previously (12), which suggested that MAG2 would facilitate a thinning of the membrane such that PGLa would be able to insert into it, cannot be correct. We can instead conclude that only lipid systems that encourage peptide insertion per se show the MAG2-induced I-state of PGLa. The relationship between lipid shape and the tendency of peptides to insert into the membrane, as previously discussed (13), is illustrated in Fig. S4. Interestingly, common bacterial lipids like PE and CL have a negative spontaneous curvature and should thus not support peptide insertion and stable pores. However, pores could still be transient in native membranes, or other components like membrane proteins could influence the overall spontaneous curvature.In conclusion, we propose several criteria that encourage a peptide to insert from the surface-bound S-state more deeply into the membrane (i.e., into a T-state or I-state): 1), positive lipid spontaneous curvature, which is enhanced by large headgroups and ordered lipid chains (due to saturation, but also found at low temperatures close to the gel-to-liquid-crystalline phase transition); 2), a narrow polar sector and uncharged termini of the peptide; and 3), the presence of another peptide. The other peptide might have an indirect effect by changing the membrane properties via crowding. However, for PGLa/MAG2, a distinct synergistic activity has been demonstrated, indicating more specific interactions between these two peptides. The present ¹⁵N-NMR analysis shows that the two partner peptides are not aligned side-by-side as a dimer. Further solid-state NMR distance measurements will be required to clarify their detailed mode of assembly.     

β-Diketones in Rhododendron waxes

Chromatographic, mass spectrometric and spectroscopic evidence has been obtained for four β-diketones occurring in the leaf waxes of some members of     

Lipid and Peptide Dynamics in Membranes upon Insertion of n-alkyl-β-D-Glucopyranosides

The effect of nonionic detergents of the n-alkyl-β-D-glucopyranoside class on the ordering of lipid bilayers and the dynamics of membrane-embedded peptides were investigated with ²H- and ³¹P-NMR. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was selectively deuterated at methylene segments C-2, C-7, and C-16 of the two fatty acyl chains. Two trans-membrane helices, WALP-19 and glycophorin A_71-98, were synthesized with Ala-d₃ in the central region of the α-helix. n-Alkyl-β-D-glucopyranosides with alkyl chains with 6, 7, 8, and 10 carbon atoms were added at increasing concentrations to the lipid membrane. The bilayer structure is retained up to a detergent/lipid molar ratio of 1:1. The insertion of the detergents leads to a selective disordering of the lipids. The headgroup region remains largely unaffected; the fatty acyl chain segments parallel to the detergent alkyl chain are only modestly disordered (10-20%), whereas lipid segments beyond the methyl terminus of the detergent show a decrease of up to 50%. The change in the bilayer order profile corresponds to an increase in bilayer entropy. Insertion of detergents into the lipid bilayers is completely entropy-driven. The entropy change accompanying lipid disorder is equivalent in magnitude to the hydrophobic effect. Ala-d₃ deuterated WALP-19 and GlycA_71-97 were incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine at a peptide/lipid molar ratio of 1:100 and measured above the 1,2-dimyristoyl-sn-glycero-3-phosphocholine gel/liquid-crystal phase transition. Well-resolved ²H-NMR quadrupole splittings were observed for the two trans-membrane helices, revealing a rapid rotation of the CD₃ methyl rotor superimposed on an additional rotation of the whole peptide around the bilayer normal. The presence of detergent fluidizes the membrane and produces magnetic alignment of bilayer domains but does not produce essential changes in the peptide conformation or dynamics.     

Cholesterol Modulates the Dimer Interface of the β2-Adrenergic Receptor via Cholesterol Occupancy Sites

The β₂-adrenergic receptor is an important member of the G-protein-coupled receptor (GPCR) superfamily, whose stability and function are modulated by membrane cholesterol. The recent high-resolution crystal structure of the β₂-adrenergic receptor revealed the presence of possible cholesterol-binding sites in the receptor. However, the functional relevance of cholesterol binding to the receptor remains unexplored. We used MARTINI coarse-grained molecular-dynamics simulations to explore dimerization of the β₂-adrenergic receptor in lipid bilayers containing cholesterol. A novel (to our knowledge) aspect of our results is that receptor dimerization is modulated by membrane cholesterol. We show that cholesterol binds to transmembrane helix IV, and cholesterol occupancy at this site restricts its involvement at the dimer interface. With increasing cholesterol concentration, an increased presence of transmembrane helices I and II, but a reduced presence of transmembrane helix IV, is observed at the dimer interface. To our knowledge, this study is one of the first to explore the correlation between cholesterol occupancy and GPCR organization. Our results indicate that dimer plasticity is relevant not just as an organizational principle but also as a subtle regulatory principle for GPCR function. We believe these results constitute an important step toward designing better drugs for GPCR dimer targets.