Order parameters and areas in fluid-phase oriented lipid membranes using wide angle X-ray scattering

We used wide angle x-ray scattering (WAXS) from stacks of oriented lipid bilayers to measure chain orientational order parameters and lipid areas in model membranes consisting of mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol in fluid phases. The addition of 40% cholesterol to either DOPC or DPPC changes the WAXS pattern due to an increase in acyl chain orientational order, which is one of the main properties distinguishing the cholesterol-rich liquid-ordered (Lo) phase from the liquid-disordered (Ld) phase. In contrast, powder x-ray data from multilamellar vesicles does not yield information about orientational order, and the scattering from the Lo and Ld phases looks similar. An analytical model to describe the relationship between the chain orientational distribution and WAXS data was used to obtain an average orientational order parameter, S_x-ray. When 40% cholesterol is added to either DOPC or DPPC, S_x-ray more than doubles, consistent with previous NMR order parameter measurements. By combining information about the average chain orientation with the chain-chain correlation spacing, we extended a commonly used method for calculating areas for gel-phase lipids to fluid-phase lipids and obtained agreement to within 5% of literature values.     

Structure and Dynamics of the Myristoyl Lipid Modification of Src Peptides Determined by H Solid-State NMR Spectroscopy

Lipid modifications of proteins are widespread in nature and play an important role in numerous biological processes. The nonreceptor tyrosine kinase Src is equipped with an N-terminal myristoyl chain and a cluster of basic amino acids for the stable membrane association of the protein. We used ²H NMR spectroscopy to investigate the structure and dynamics of the myristoyl chain of myr-Src(2-19), and compare them with the hydrocarbon chains of the surrounding phospholipids in bilayers of varying surface potentials and chain lengths. The myristoyl chain of Src was well inserted in all bilayers investigated. In zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine membranes, the myristoyl chain of Src was significantly longer and appears “stiffer” than the phospholipid chains. This can be explained by an equilibrium between the attraction attributable to the insertion of the myristoyl chain and the Born repulsion. In a 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine] membrane, where attractive electrostatic interactions come into play, the differences between the peptide and the phospholipid chain lengths were attenuated, and the molecular dynamics of all lipid chains were similar. In a much thicker 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine]/cholesterol membrane, the length of the myristoyl chain of Src was elongated nearly to its maximum, and the order parameters of the Src chain were comparable to those of the surrounding membrane.     

Melatonin Alters Fluid Phase Coexistence in POPC/DPPC/Cholesterol Membranes

The structure and biophysical properties of lipid membranes are important for cellular functions in health and disease. In Alzheimer’s disease, the neuronal membrane is a target for toxic amyloid-β (Aβ). Melatonin is an important pineal gland hormone that has been shown to protect against Aβ toxicity in cellular and animal studies, but the molecular mechanism of this protection is not fully understood. Melatonin is a small membrane-active molecule that has been shown to interact with model lipid membranes and alter the membrane biophysical properties, such as membrane molecular order and dynamics. This effect of melatonin has been previously studied in simple model bilayers with one or two lipid components. To make it more relevant to neuronal membranes, we used a more complex ternary lipid mixture as our membrane model. In this study, we used ²H-NMR to investigate the effect of melatonin on the phase behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and cholesterol lipid membranes. We used deuterium-labeled POPC-d₃₁ and DPPC-d₆₂,separately to probe the changes in hydrocarbon chain order as a function of temperature and melatonin concentration. We find that POPC/DPPC/cholesterol at molar proportions of 3:3:2 is close to liquid-disordered/liquid-ordered phase separation and that melatonin can induce phase separation in these ternary mixtures by preferentially incorporating into the disordered phase and increasing its level of disorder. At 5 mol% melatonin, we observed phase separation in samples with POPC-d₃₁, but not with DPPC-d₆₂, whereas at 10 mol% melatonin, phase separation was observed in both samples with either POPC-d₃₁ or DPPC-d₆₂. These results indicate that melatonin can have a strong effect on membrane structure and physical properties, which may provide some clues to understanding how melatonin protects against Aβ, and that choice of chain perdeuteration is an important consideration from a technical point of view.     

Characterization of the binary mixed monolayers of α-tocopherol with phospholipids at the air-water interface

The mixed Langmuir monolayers composed of model constituents of biological membranes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), were investigated to provide information on the intermolecular interactions between these membrane components and the physiologically active vitamin E–α-tocopherol (TF), as well as on the phase behavior of these mixed systems. Additionally, topography of these monolayers transferred onto the mica support was investigated by the inverted metallurgical microscope. Morphological characteristics were directly observed by Brewster angle microscopy (BAM). From the surface pressure–area isotherms and the analysis of physicochemical parameters (compressibility and mean molecular area at the maximum compressibility) it was found that depending on the acyl chains saturation degree, TF has different effect on the phospholipids. In the case of DPPC, the addition of TF to the phospholipid film causes destabilization of the ordered hydrocarbon chains, while in the POPC/DOPC–TF systems, the attractive interactions are responsible for the monolayer increased stability. Thus, the results of these studies confirm the hypothesis that α-tocopherol may play a role in the stabilization of biological membranes.     

Membrane properties of plant sterols in phospholipid bilayers as determined by differential scanning calorimetry, resonance energy transfer and detergent-induced solubilization

The increased use of plant sterols as cholesterol-lowering agents warrants further research on the possible effects of plant sterols in membranes. In this study, the effects of the incorporation of cholesterol, campesterol, β-sitosterol and stigmasterol in phospholipid bilayers were investigated by differential scanning calorimetry (DSC), resonance energy transfer (RET) between trans parinaric acid (tPA) and 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-PC), and Triton X-100-induced solubilization. The phospholipids used were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), d-erythro-N-palmitoyl-sphingomyelin (PSM), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). In DSC experiments, it was demonstrated that the sterols differed in their effect on the melting temperatures of both the sterol-poor and the sterol-rich domains in DPPC and PSM bilayers. The plant sterols gave rise to lower temperatures of both transitions, when compared with cholesterol. The plant sterols also resulted in lower transition temperatures, in comparison with cholesterol, when sterol-containing DPPC and PSM bilayers were investigated by RET. In the detergent solubilization experiments, the total molar ratio between Triton X-100 and POPC at the onset of solubilization (R_t,sat) was higher for bilayers containing plant sterols, in comparison with membranes containing cholesterol. Taken together, the observations presented in this study indicate that campesterol, β-sitosterol and stigmasterol interacted less favorably than cholesterol with the phospholipids, leading to measurable differences in their domain properties.     

Alternative Mechanisms for the Interaction of the Cell-Penetrating Peptides Penetratin and the TAT Peptide with Lipid Bilayers

Cell-penetrating peptides (CPPs) have recently attracted much interest due to their apparent ability to penetrate cell membranes in an energy-independent manner. Here molecular-dynamics simulation techniques were used to study the interaction of two CPPs: penetratin and the TAT peptide with 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) phospolipid bilayers shed light on alternative mechanisms by which these peptides might cross biological membranes. In contrast to previous simulation studies of charged peptides interacting with lipid bilayers, no spontaneous formation of transmembrane pores was observed. Instead, the simulations suggest that the peptides may enter the cell by micropinocytosis, whereby the peptides induce curvature in the membrane, ultimately leading to the formation of small vesicles within the cell that encapsulate the peptides. Specifically, multiple peptides were observed to induce large deformations in the lipid bilayer that persisted throughout the timescale of the simulations (hundreds of nanoseconds). Pore formation could be induced in simulations in which an external potential was used to pull a single penetratin or TAT peptide into the membrane. With the use of umbrella-sampling techniques, the free energy of inserting a single penetratin peptide into a DPPC bilayer was estimated to be ∼75 kJmol⁻¹, which suggests that the spontaneous penetration of single peptides would require a timescale of at least seconds to minutes. This work also illustrates the extent to which the results of such simulations can depend on the initial conditions, the extent of equilibration, the size of the system, and the conditions under which the simulations are performed. The implications of this with respect to the current systems and to simulations of membrane-peptide interactions in general are discussed.     

Lipid Organization in Mixed Lipid Membranes Driven by Intrinsic Curvature Difference

Laurdan fluorescence, novel spectral fitting, and dynamic light scattering were combined to determine lateral lipid organization in mixed lipid membranes of the oxidized lipid, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC), and each of the three bilayer lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). Second harmonic spectra were computed to determine the number of elementary emissions present. All mixtures indicated two emissions. Accordingly, spectra were fit to two log-normal distributions. Changes with PGPC mole fraction, X_PGPC, of the area of the shorter wavelength line and of dynamic light scattering-derived aggregate sizes show that: DPPC and PGPC form component-separated mixed vesicles for X_PGPC ≤ 0.2 and coexisting vesicles and micelles for X_PGPC > 0.2 in gel and liquid-ordered phases and for all X_PGPC in the liquid-disordered phase; POPC and PGPC form randomly mixed vesicles for X_PGPC ≤ 0.2 and component-separated mixed vesicles for X_PGPC > 0.2. DOPC and PGPC separate into vesicles and micelles. Component segregation is due to unstable inhomogeneous membrane curvature stemming from lipid-specific intrinsic curvature differences between mixing molecules. PGPC is inverse cone-shaped because its truncated tail with a terminal polar group points into the interface. It is similar to and mixes with POPC, also an inverse cone because of mobility of its unsaturated tail. PGPC is least similar to DOPC because mobilities of both unsaturated tails confer a cone shape to DOPC, and PGPC separates form DOPC. DPPC and PGPC do not mix in the liquid-disordered phase because mobility of both tails in this phase renders DPPC a cone. DPPC is a cylinder in the gel phase and of moderate similarity to PGPC and mixes moderately with PGPC.     

Permeability of DOPC bilayers under photoinduced oxidation: Sensitivity to photosensitizer

The modification of lipid bilayer permeability is one of the most striking yet poorly understood physical transformations that follow photoinduced lipid oxidation. We have recently proposed that the increase of permeability of photooxidized 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers is controlled by the time required by the oxidized lipid species to diffuse and aggregate into pores. Here we further probe this mechanism by studying photosensitization of DOPC membranes by methylene blue (MB) and DO15, a more hydrophobic phenothiazinium photosensitizer, under different irradiation powers. Our results not only reveal the interplay between the production rate and the diffusion of the oxidized lipids, but highlight also the importance of photosensitizer localization in the kinetics of oxidized membrane permeability.     

Phase Separation in Lipid Membranes

Cell membranes show complex behavior, in part because of the large number of different components that interact with each other in different ways. One aspect of this complex behavior is lateral organization of components on a range of spatial scales. We found that lipid-only mixtures can model the range of size scales, from approximately 2 nm up to microns. Furthermore, the size of compositional heterogeneities can be controlled entirely by lipid composition for mixtures such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol or sphingomyelin (SM)/DOPC/POPC/cholesterol. In one region of special interest, because of its connection to cell membrane rafts, nanometer-scale domains of liquid-disordered phase and liquid-ordered phase coexist over a wide range of compositions.     

Specific RNA binding to ordered phospholipid bilayers

We have studied RNA binding to vesicles bounded by ordered and disordered phospholipid membranes. A positive correlation exists between bilayer order and RNA affinity. In particular, structure-dependent RNA binding appears for rafted (liquid-ordered) domains in sphingomyelin-cholesterol-1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. Binding to more highly ordered gel phase membranes is stronger, but much less RNA structure-dependent. All modes of RNA-membrane association seem to be electrostatic and headgroup directed. Fluorometry on 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes indicates that bound RNA broadens the gel-fluid melting transition, and reduces lipid headgroup order, as detected via fluorometric measurement of intramembrane electric fields. RNA preference for rafted lipid was visualized and confirmed using multiple fluorophores that allow fluorescence and fluorescence resonance energy transfer microscopy on RNA molecules closely associated with ordered lipid patches within giant vesicles. Accordingly, both RNA structure and membrane order could modulate biological RNA–membrane interactions.     

Domain nucleation rates and interfacial line tensions in supported bilayers of ternary mixtures containing galactosylceramide

Domains within the plane of the plasma membrane, referred to as membrane rafts, have been a topic of considerable interest in the field of membrane biophysics. Although model membrane systems have been used extensively to study lipid phase behavior as it relates to the existence of rafts, very little work has focused on either the initial stage of lipid domain nucleation, or the relevant physical parameters such as temperature and interfacial line tension which control nucleation. In this work, we utilize a method in which the kinetic process of lipid domain nucleation is imaged by atomic force microscopy and modeled using classical theory of nucleation to map interfacial line tension in ternary lipid mixtures. These mixtures consist of a fluid phase lipid component (1,2-dilauroyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphocholine), a solid phase component (galactosylceramide), and cholesterol. Interfacial line tension measurements of galactosylceramide-rich domains track with our previously measured area/perimeter ratios and height mismatches measured here. Line tension also follows known trends in cholesterol interactions and partitioning, as we observed previously with area/perimeter ratios. Our line tension measurements are discussed in combination with recent line tension measurements to address line tension regulation by cholesterol and the dynamic nature of membrane rafts.     

Perturbation of DPPC bilayers by high concentrations of pulmonary surfactant protein SP-B

Deuterium (²H) NMR has been used to observe perturbation of dipalmitoylphosphatidylcholine (DPPC) bilayers by the pulmonary surfactant protein B (SP-B) at concentrations up to 17% (w/w). Previous ²H NMR studies of DPPC/dipalmitoylphosphatidylglycerol (DPPG) (7:3) bilayers containing up to 11% (w/w) SP-B and DPPC bilayers containing up to 11% (w/w) synthetic SP-B indicated a slight effect on bilayer chain order and a more substantial effect on motions that contribute to decay of quadrupole echoes obtained from bilayers of deuterated DPPC. This is consistent with the perturbation of headgroup-deuterated DPPC reported here for bilayers containing 6 and 9% (w/w) SP-B. For the higher concentrations of SP-B investigated in the present work, ²H NMR spectra of DPPC deuterated in both the headgroup and chain display a prominent narrow component consistent with fast, large amplitude reorientation of some labeled lipid. Similar spectral perturbations have been reported for bilayers in the presence of the antibiotic polypeptide nisin. The observation of large amplitude lipid reorientation at high SP-B concentration could indicate that SP-B can induce regions of high bilayer curvature and thus provides some insight into local interaction of SP-B with DPPC. Such local interactions may be relevant to the formation, in vitro and in vivo, of tubular myelin, a unique structure found in extracellular pulmonary surfactant, and to the delivery of surfactant material to films at the air–water interface.Abbreviations DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine - DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol - DPPC-d₆₂ 1,2-perdeuterodipalmitoyl-sn-glycero-3-phosphocholine - DPPC-d₄ 1,2-dipalmitoyl-sn-glycero-3-phospho-(, perdeutero)-choline     

Effect of Membrane Composition on Antimicrobial Peptides Aurein 2.2 and 2.3 From Australian Southern Bell Frogs

The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an α-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The ³¹P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC₃5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.     

Investigation of phase transitions of saturated phosphocholine lipid bilayers via molecular dynamics simulations

Lipid bilayers play an important role in biological systems as they protect cells against unwanted chemicals and provide a barrier for material inside a cell from leaking out. In this paper, nearly 30 μs of molecular dynamics (MD) simulations were performed to investigate phase transitions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-phosphocholine (DPPC) lipid bilayers from the liquid crystalline (L_α) to the ripple (P_β) and to the gel phase (L_β). Our MD simulations accurately predict the main transition temperature for the single-component bilayers. A key focus of this work is to quantify the structure of the P_β phase for DMPC and compare with measures from x-ray experiments. The P_β major arm has similar structure to that of the L_β, while the thinner minor arm has interdigitated chains and the transition region between these two regions has large chain splay and disorder. At lower temperatures, our MD simulations predict the formation of the L_β phase with tilted fatty acid chains. The P_β and L_β phases are studied for mixtures of DMPC and DPPC and compare favorably with experiment. Overall, our MD simulations provide evidence for the relevancy of the CHARMM36 lipid force field for structures and add to our understanding of the less-defined P_β phase.     

Influence of lipid membrane rigidity on properties of supporting polymer

Temperature-sensitive hydrogel polymers are utilized as responsive layers in various applications. Although the polymer's native characteristics have been studied extensively, details concerning its properties during interaction with biorelated structures are lacking. This work investigates the interaction between a thermoresponsive polymer cushion and different lipid membrane capping layers probed by neutron reflectometry. N-isopropylacrylamide copolymerized with methacroylbenzophenone first supported a lipid bilayer composed of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and subsequently 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The polymer-membrane systems were investigated above and below the polymer transition temperature (37 and 25°C). Although the same cushion supported each lipid membrane, the polymer hydration profile and thickness were markedly different for DPPE and DPPC systems. Because DPPE and DPPC have different bending rigidities, these results establish that the polymer-membrane interaction is critically mediated by the mechanics of the membrane, providing better insight into cell-hydrogel interactions.     

Assembly of the M2 Tetramer Is Strongly Modulated by Lipid Chain Length

The influenza virus matrix protein 2 (M2) assembles into a tetramer in the host membrane during viral uncoating and maturation. It has been used as a model system to understand the relative contributions of protein-lipid and protein-protein interactions to membrane protein structure and association. Here we investigate the effect of lipid chain length on the association of the M2 transmembrane domain into tetramers using Förster resonance energy transfer. We observe that the interactions between the M2 helices are much stronger in 1,2-dilauroyl-sn-glycero-3-phosphocholine than in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. Thus, lipid chain length and bilayer thickness not only modulate peptide interactions, but could also be a major determinant of the association of transmembrane helices into functional membrane protein oligomers.     

In-plane miscibility and mixed bilayer microstructure in mixtures of catanionic glycolipids and zwitterionic phospholipids

SAXS/WAXS studies were performed in combination with freeze fracture electron microscopy using mixtures of a new Gemini catanionic surfactant (Gem16-12, formed by two sugar groups bound by a hydrocarbon spacer with 12 carbons and two 16-carbon chains) and the zwitterionic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) to establish the phase diagram. Gem16-12 in water forms bilayers with the same amount of hydration water as DPPC. A frozen interdigitated phase with a low hydration number is observed below room temperature. The kinetics of the formation of this crystalline phase is very slow. Above the chain melting temperature, multilayered vesicles are formed. Mixing with DPPC produces mixed bilayers above the corresponding chain melting temperature. At room temperature, partially lamellar aggregates with local nematic order are observed. Splitting of infinite lamellae into discs is linked to immiscibility in frozen state. The ordering process is always accompanied by dehydration of the system. As a consequence, an unusual order-disorder phase transition upon cooling is observed.     

The influence of NBD fluorescent probe on model membranes containing POPC and DPPC

To investigate the effect of fluorescent probe on the properties of membranes, we studied model membranes composed of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl 2-oleoyl-sn-glycero-3-phosphocholine (POPC) in the presence and absence of fluorescent probe. The morphology of giant unilamellar vesicles (GUVs) has been observed as a function of temperature and composition by fluorescence microscopy using NBD-DOPE or C₆-NBD-PC as the probe. The phase behavior of model membranes containing no fluorescent probe was investigated by ²H-NMR spectroscopy. We found that the bright phase observed on GUVs was the fluid phase enriched in POPC and the dark phase was the gel phase enriched in DPPC. NBD-DOPE and C₆-NBD-PC preferentially participated in the fluid-phase domains when GUVs were in the gel?+?fluid phase coexistence. Inclusion of both fluorescent probes (1?mol%) lowered the transition temperature of POPC/DPPC membranes. In addition, C₆-NBD-PC exhibited a stronger effect than NBD-DOPE, which was considered to be associated with the structures of fluorescent molecules.     

Amyloidogenic Propensities and Conformational Properties of ProIAPP and IAPP in the Presence of Lipid Bilayer Membranes

Human islet amyloid polypeptide (hIAPP), which is considered the primary culprit for β-cell loss in type 2 diabetes mellitus patients, is synthesized in β-cells of the pancreas from its precursor pro-islet amyloid polypeptide (proIAPP), which may be important in early intracellular amyloid formation as well. We compare the amyloidogenic propensities and conformational properties of proIAPP and hIAPP in the presence of negatively charged lipid membranes, which have been discussed as loci of initiation of the fibrillation reaction. Circular dichroism studies verify the initial secondary structures of proIAPP and hIAPP to be predominantly unordered with small amounts of ordered secondary structure elements, and exhibit minor differences between these two peptides only. Using attenuated total reflection-Fourier transform infrared spectroscopy and thioflavin T fluorescence spectroscopy, as well as atomic force microscopy, we show that in the presence of negatively charged membranes, proIAPP exhibits a much higher amyloidogenic propensity than in bulk solvent. Compared to hIAPP, it is still much less amyloidogenic, however. Although differences in the secondary structures of the aggregated species of hIAPP and proIAPP at the lipid interface are small, they are reflected in morphological changes. Unlike hIAPP, proIAPP forms essentially oligomeric-like structures at the lipid interface. Besides the interaction with anionic membranes [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) + x1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]], interaction with zwitterionic homogeneous (DOPC) and heterogeneous (1,2-dipalmitoyl-sn-glycero-3-phosphocholine:DOPC:cholesterol 1:2:1 model raft mixture) membranes has also been studied. Both peptides do not aggregate significantly at DOPC bilayers. In the presence of the model raft membrane, hIAPP aggregates markedly as well. Conversely, proIAPP clusters into less ordered structures and to a minor extent at raft membranes only. The addition of proIAPP to hIAPP retards the hIAPP fibrillation process also in the presence of negatively charged lipid bilayers. In excess proIAPP, increased aggregation levels are finally observed, however, which could be attributed to seed-induced cofibrillation of proIAPP.     

Depolarization Laplace Transform Analysis of Exchangeable Hyperpolarized Xe for Detecting Ordering Phases and Cholesterol Content of Biomembrane Models

We present a highly sensitive nuclear-magnetic resonance technique to study membrane dynamics that combines the temporary encapsulation of spin-hyperpolarized xenon (¹²⁹Xe) atoms in cryptophane-A-monoacid (CrA_ma) and their indirect detection through chemical exchange saturation transfer. Radiofrequency-labeled Xe@CrA_ma complexes exhibit characteristic differences in chemical exchange saturation transfer-driven depolarization when interacting with binary membrane models composed of different molecular ratios of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). The method is also applied to mixtures of cholesterol and POPC. The existence of domains that fluctuate in cluster size in DPPC/POPC models at a high (75–98%) DPPC content induces up to a fivefold increase in spin depolarization time τ at 297 K. In POPC/cholesterol model membranes, the parameter τ depends linearly on the cholesterol content at 310 K and allows us to determine the cholesterol content with an accuracy of at least 5%.