Membrane interactions and the effect of metal ions of the amyloidogenic fragment Aβ(25-35) in comparison to Aβ(1-42)

Aβ(1−42) peptide, found as aggregated species in Alzheimer's disease brain, is linked to the onset of Alzheimer's disease. Many reports have linked metals to inducing Aβ aggregation and amyloid plaque formation. Aβ(25-35), a fragment from the C-terminal end of Aβ(1−42), lacks the metal coordinating sites found in the full-length peptide and is neurotoxic to cortical cortex cell cultures. We report solid-state NMR studies of Aβ(25-35) in model lipid membrane systems of anionic phospholipids and cholesterol, and compare structural changes to those of Aβ(1-42). When added after vesicle formation, Aβ(25-35) was found to interact with the lipid headgroups and slightly perturb the lipid acyl-chain region; when Aβ(25-35) was included during vesicle formation, it inserted deeper into the bilayer. While Aβ(25-35) retained the same β-sheet structure irrespective of the mode of addition, the longer Aβ(1-42) appeared to have an increase in β-sheet structure at the C-terminus when added to phospholipid liposomes after vesicle formation. Since the Aβ(25-35) fragment is also neurotoxic, the full-length peptide may have more than one pathway for toxicity.     

The influence of phospholipid membranes on bovine calcitonin secondary structure and amyloid formation

Calcitonin, a peptide hormone associated with medullary carcinoma of the thyroid, has the potential to form amyloid fibrils and may be a valuable model for investigating the role of peptide-membrane interactions in beta-sheet and amyloid formation. Via a new model peptide system, bovine calcitonin, we found that the exposure of peptide to phospholipid membranes altered its structure relative to the structures formed in aqueous solutions. Of particular relevance to the amyloidoses, incubation of calcitonin with cholesterol-rich and ganglioside-containing membranes resulted in significant enrichment in the beta-sheet and amyloid content of the peptide. The formation of amyloid was also accelerated in these systems. A correlation between the phospholipid-induced structural alterations and calcitonin binding affinities to phospholipid membranes was evident. Bovine calcitonin has considerably higher binding affinity for the phospholipid systems that enhanced its beta-sheet and amyloid structure. Electrostatic forces were not the governing forces behind the observed behavior, as supported by the fact that the ionic strength did not affect the peptide structures or binding affinities. A Van't Hoff analysis of the temperature-dependent peptide binding affinities indicated that binding led to an increase in enthalpy and possibly an increase in entropy of the peptide-membrane systems. Experiments with other amyloid-forming peptides such as beta-amyloid of Alzheimer's disease have also shown similar results and may indicate the need to manipulate peptide-membrane interactions in order to control amyloid formation and its associated disease.     

Resolving the kinetics of lipid,protein and peptide diffusion in membranes

Abstract

Recent developments in the understanding of molecular diffusion phenomena in membranes are reviewed. Both model bilayers and biological membranes are considered in respect of lateral diffusion, rotational diffusion and transverse diffusion (flip-flop). For model systems, particular attention is paid to recent data obtained using surface-specific techniques such as sum frequency generation vibrational spectroscopy on supported lipid bilayers, and fluorescence correlation spectroscopy on giant unilamellar vesicles, both of which have yielded new insights into the intrinsic rates of diffusion and the energetic barriers to processes such as lipid flip-flop. Advances in single-molecule and many-molecule fluorescence methodologies have enabled the observation of processes such as anomalous diffusion for some membrane species in biological membranes. These are discussed in terms of new models for the role of membrane interactions with the cytoskeleton, the effects of molecular crowding in membranes, and the formation of lipid rafts. The diffusion of peptides, proteins and lipids is considered, particularly in relation to the means by which antimicrobial peptide activity may be rationalized in terms of membrane poration and lipid flip-flop.     

Interactions of the channel forming peptide alamethicin with artificial and natural membranes

Alamethicin and related α-aminoisobutyric acid peptides form transmembrane channels across lipid bilayers. This article briefly reviews studies on the effect of alamethicin on lipid phase transitions in lipid bilayers and on mitochondrial oxidative phosphorylation. Fluorescence polarization studies, employing 1,6-diphenyl-1,3,5-hexatriene as a probe, suggest that alamethicin fluidizes lipid bilayers below the phase transition t-emperature, but has little effect above the gel-liquid crystal transition point. Alamethicin is shown to function as an uncoupler of oxidative phosphorylation in rat liver mitochondria. The influence of alamethicin on mitochondrial respiration is modulated by the phosphate ion concentration in the medium. Classical uncoupling activity is evident at low phosphate levels while inhibitory effects set in at higher phosphate concentrations. Time-dependent changes in respiration rates following peptide addition are rationalized in terms of alamethicin interactions with mitochondrial membrane components.     

13C, 15N Resonance Assignment of Parts of the HET-s Prion Protein in its Amyloid Form

The partial ¹⁵N and ¹³C solid-state NMR resonance assignment of the HET-s prion protein fragment 218–289 in its amyloid form is presented. It is based on experiments measured at MAS frequencies in the range of 20–40 kHz using exclusively adiabatic polarization-transfer schemes. The resonance assignment within each residue is based on two-dimensional ¹³C––¹³C correlation spectra utilizing the DREAM mixing scheme. The sequential linking of the assigned residues used a set of two- and three-dimensional ¹⁵N––¹³C correlation experiments. Almost all cross peaks visible in the spectra are assigned, but only resonances from 43 of the 78 amino-acid residues could be detected. The missing residues are thought to be highly disordered and/or highly dynamic giving rise to broad resonance lines that escaped detection in the experiments applied. The line widths of the observed resonances are narrow and comparable to line widths observed in micro-crystalline samples. The 43 assigned residues are located in two fragments of about 20 residues. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Insertion of peptide chains into lipid membranes: An off-lattice Monte Carlo dynamics model

A combination of dynamic Monte Carlo simulation techniques with a hydropathy scale method for the prediction of the location of transmembrane fragments in membrane proteins is described. The new hydropathy scale proposed here is based on experimental data for the interactions of tripeptides with phospholipid membranes (Jacobs, R.E., White, S.H. Biochemistry 26:6127–6134, 1987) and the self-solvation effect in protein systems (Roseman, M.A., J. Mol. Biol. 200:513–522, 1988). The simulations give good predictions both for the state of association and the orientation of the peptide relative to the membrane surface of a number of peptides including Magainin2, M2δ, and melittin. Furthermore, for Pf1 bacterio-phage coat protein, in accord with experiment, the simulations predict that the C-terminus forms a transmembrane helix and the N-terminus forms a helix which is adsorbed on the surface of the bilayer. Finally, the present series of simulations provide a number of insights into the mechanism of insertion of peptides into cell membranes. © 1993 Wiley-Liss, Inc.     

Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy.

Magainin 2 is a 23-residue peptide that forms an amphipathic alpha-helix in membrane environments. It functions as an antibiotic and is known to disrupt the electrochemical gradients across the cell membranes of many bacteria, fungi, and some tumor cells, although it does not lyse red blood cells. One- and two-dimensional solid-state 15N NMR spectra of specifically 15N-labeled magainin 2 in oriented bilayer samples show that the secondary structure of essentially the entire peptide is alpha-helix, immobilized by its interactions with the phospholipids, and oriented parallel to the membrane surface.     

Orientational constraints as three-dimensional structural constraints from chemical shift anisotropy: the polypeptide backbone of gramicidin A in a lipid bilayer.

Chemical shifts observed from samples that are uniformly aligned with respect to the magnetic field can be used as very high-resolution structural constraints. This constraint takes the form of an orientational constraint rather than the more familiar distance constraint. The accuracy of these constraints is dependent upon the quality of the tensor characterization. Both tensor element magnitudes and tensor orientations with respect to the molecular frame need to be considered. Here these constraints have been used to evaluate models for the channel conformation of gramicidin A. Of the three models used, the one experimentally derived model of gramicidin in sodium dodecyl sulfate micelles fits the data least well.     

Role of some trace metal ions in placental membrane lipid peroxidation

The antioxidative and/or pro-oxidative potential of three trace metal ions, namely aluminum (Al), manganese (Mn), and selenium (Se), has been studied. The effect of Al and Mn was found to be anion independent. The pro-oxidative potential of Al was more prominent than its antioxidative potential. This may be due to its redox inert nature. The increase in lipid peroxidation rates in placental syncytiotroblast membranes may contribute to the etiology of aluminum toxicity. Selenium had an antioxidative potential only in the whole-cell homogenate. This appears to be mediated by glutathione peroxidase of which Se is a cofactor. Manganese proved to be the trace metal ion of choice. It decreased the production of thiobarbituric acid reactive substances (TBARS). This may be due to its capacity to quench the superoxide anion and hydroxyl radicals and also due to its chain-breaking capacity. During the present course, ferrous-ascorbate mediated lipid peroxidation has been studied using various combinations of FeSO₄ and ascorbic acid. Extrapolating the combined ratio of the individual combination as substrate concentration ([S]) and treating the observed amount of malondialdehyde (MDA) produced equivalent to initial velocity (v _i ), as in the case of enzymatic studies, the data were treated according to Michaelis-Menten kinetics and the values of k _c and C _max have been calculated.     

Binding of prion protein to lipid membranes and implications for prion conversion

The binding of the Syrian hamster prion protein, SHaPrP(90-231), to model lipid membranes was investigated by tryptophan fluorescence. Membranes composed of negatively charged or zwitterionic lipids, and raft-like membranes containing dipalmitoylphosphatidylcholine(1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol and sphingomyelin, were investigated. It was found that SHaPrP(90-231) binds to negatively charged lipid membranes and raft-like membranes. Binding of PrP to negatively charged lipid membranes involves both electrostatic and hydrophobic lipid-protein interactions and results in partial insertion of PrP into the lipid bilayer. This membrane-inserted conformation of PrP is richer in beta-sheet structure and has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents. In contrast, the binding of PrP to raft-like membranes is driven by hydrophobic lipid-protein interactions and induces the formation of alpha-helical structure. This conformation of PrP with a high content of alpha-helix is formed only at pH 7 and does not destabilize the lipid bilayer. Our findings support the view that an interaction of PrP with lipid membranes could play a role in PrP conversion.     

Structural Mechanism of the Interaction of Alzheimer Disease Aβ Fibrils with the Non-steroidal Anti-inflammatory Drug (NSAID) Sulindac Sulfide

Alzheimer disease is the most severe neurodegenerative disease worldwide. In the past years, a plethora of small molecules interfering with amyloid-β (Aβ) aggregation has been reported. However, their mode of interaction with amyloid fibers is not understood. Non-steroidal anti-inflammatory drugs (NSAIDs) are known γ-secretase modulators; they influence Aβ populations. It has been suggested that NSAIDs are pleiotrophic and can interact with more than one pathomechanism. Here we present a magic angle spinning solid-state NMR study demonstrating that the NSAID sulindac sulfide interacts specifically with Alzheimer disease Aβ fibrils. We find that sulindac sulfide does not induce drastic architectural changes in the fibrillar structure but intercalates between the two β-strands of the amyloid fibril and binds to hydrophobic cavities, which are found consistently in all analyzed structures. The characteristic Asp²³-Lys²⁸ salt bridge is not affected upon interacting with sulindac sulfide. The primary binding site is located in the vicinity of residue Gly³³, a residue involved in Met³⁵ oxidation. The results presented here will assist the search for pharmacologically active molecules that can potentially be employed as lead structures to guide the design of small molecules for the treatment of Alzheimer disease.     

A Langmuir film approach to elucidating interactions in lipid membranes: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine/cholesterol/metal cation systems

The interactions between two membrane lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and cholesterol (CHOL), were studied in Langmuir films using surface pressure isotherms and Brewster angle microscopy. The DPPE/CHOL interactions were probed for chosen monolayer and subphase (Na(+), Ca(2+)) composition at 20, 25, and 30 degrees C. The results obtained show that DPPE and CHOL are miscible for the cholesterol mol fractions x(CHOL)=0.3-0.5. Cholesterol induces condensation of the DPPE monolayers. The most significant condensation of the DPPE/CHOL monolayers was observed in the presence of Ca(2+) ions in the subphase at x(CHOL)=0.4. The negative deviation of the molecular surface area (MMA) additivity from the ideal behavior together with negative values of excess free enthalpy of mixing in the monolayers were interpreted in terms of attractive interactions between lipid molecules.     

Interaction of the fusogenic peptide B18 in its amyloid-state with lipid membranes studied by solid state NMR

The interaction of the fusogenic polypeptide segment "B18" from the fertilization protein binding with lipid membranes was investigated by solid state 2H and 31P NMR, and by differential scanning calorimetry. B18 is known to adopt different conformations depending on peptide concentration, ionic conditions, pH and lipid environment. Here, the peptide was studied in its beta-stranded amyloid conformation. According to 31P NMR, the lamellar morphology of the DMPC bilayer remains intact in the presence of B18. In going from low (1:90) to high (1:10) peptide/lipid ratios, an increasing effect on several different 2H-labeled lipid segments was observed, reflecting changes in phase behavior and local dynamics. The strongest influence of B18 was detected at the acyl-chains, while no significant effect on the lipid headgroup conformation was observed. This suggests an insertion of B18 in its fibrillar state into the membrane driven by hydrophobic interactions, rather than a peripheral binding mediated by electrostatics.     

Solid-state NMR studies of the membrane-bound closed state of the colicin E1 channel domain in lipid bilayers.

The colicin E1 channel polypeptide was shown to be organized anisotropically in membranes by solid-state NMR analysis of samples of uniformly 15N-labeled protein in oriented planar phospholipid bilayers. The 190 residue C-terminal colicin E1 channel domain is the largest polypeptide to have been characterized by 15N solid-state NMR spectroscopy in oriented membrane bilayers. The 15N-NMR spectra of the colicin E1 show that: (1) the structure and dynamics are independent of anionic lipid content in both oriented and unoriented samples; (2) assuming the secondary structure of the polypeptide is helical, there are both trans-membrane and in-plane helical segments; (3) trans-membrane helices account for approximately 20-25% of the channel polypeptide, which is equivalent to 38-48 residues of the 190-residue polypeptide. The results of the two-dimensional PISEMA spectrum are interpreted in terms of a single trans-membrane helical hairpin inserted into the bilayer from each channel molecule. These data are also consistent with this helical hairpin being derived from the 38-residue hydrophobic segment near the C-terminus of the colicin E1 channel polypeptide.     

Transport of mercury compounds across bimolecular lipid membranes: effect of lipid composition, pH and chloride concentration

The use of bimolecular lipid membranes (BLM) as model membrane allows the analysis of the transport of mercury compounds across the lipidic barriers of biological membranes. The results of flux measurements show that two mercury compounds--HgCl2 and CH3HgCl--cross the BLM but the overall permeabilities are dependent on the pH of the aqueous media, and are not apparently influenced by the different phospholipid constituents of the bilayers. On the other hand, electrical measurements show that, function of the chemical speciation, the transport of this metal is done essentially in the neutral form.     

Interactions between biliary lipid micelles and intestinal brush border membranes investigated by 1H and 31P nuclear magnetic resonance

The effect of taurocholate and lecithincholesterol-taurocholate mixed micelles on the structure of isolated intestinal brush border membranes was investigated by nuclear magnetic resonance (NMR). Rabbit brush border membranes isolated by a Mg²⁺ precipitation step were chosen for this study because of their stability and integrity as revealed by ³¹P NMR. Incubation of taurocholate with the brush border membranes does not induce significant solubilization of these membranes even when the taurocholate/phospholipid ratio reaches 3.0 ¹H NMR studies indicate that taurocholate is included in the membrane bilayer at low concentration (3 mM). However this biliary salt produces a size diminution of the vesicles when its concentration increases. Incorporation of lecithin or lecithin-cholesterol in micelles of taurocholate and subsequent incubation with brush border membranes lead simultaneously to a decrease in the ³¹P NMR isotropic/bilayer line ratio, and to an increase in . These results indicate a protective effect of these compounds against lytic damage of taurocholate. Futhermore the equilibrium distribution of lecithin between mixed micelles and the membrane bilayer is strongly in favour of complete integration of micellar components in the bilayer. These data suggest that uptake of lipids from the micellar phase by isolated brush border membranes involves an interaction of the micelles with membranes followed by a fusion process.     

The effect of microwave radiation on the stability and formation of gramicidin-A channels in lipid bilayer membranes.

The effects of microwaves on the single-channel kinetics of gramicidin-A channels in lipid bilayer membranes were examined. Attempts were made to separate thermal and athermal effects by accurate measurements of temperature at the site of the membrane and by relating the measured parameters to their previously characterized temperature dependence. It was found that microwave radiation does not affect single-channel conductance or channel life time to a degree that is significantly different from that expected of a purely thermal effect. On the other hand, the rate of channel formation is decreased during exposure, which is opposite to that expected of a purely thermal effect. The mechanism of this effect is discussed in terms of the dimerization process of channel formation.     

beta2-microglobulin H31Y variant 3D structure highlights the protein natural propensity towards intermolecular aggregation

beta2-Microglobulin (beta2m) is the non-covalently bound light chain of the human class I major histocompatibility complex (MHC-I). The natural turnover of MHC-I gives rise to the release of beta2m into plasmatic fluids and to its catabolism in the kidney. beta2m dissociation from the heavy chain of the complex is a severe complication in patients receiving prolonged hemodialysis. As a consequence of renal failure, the increasing beta2m concentrations can lead to deposition of the protein as amyloid fibrils. Here we characterize the His31-->Tyr human beta2m mutant, a non-natural form of beta2m that is more stable than the wild-type protein, displaying a ten-fold acceleration of the slow phase of folding. We report the 2.9A resolution crystal structure and the NMR characterization of the mutant beta2m, focussing on selected structural features and on the molecular packing observed in the crystals. Juxtaposition of the four mutant beta2m molecules contained in the crystal asymmetric unit, and specific hydrogen bonds, stabilize a compact protein assembly. Conformational heterogeneity of the four independent molecules, some of their mutual interactions and partial unpairing of the N-terminal beta-strand in one protomer are in keeping with the amyloidogenic properties displayed by the mutant beta2m.