phospholipids: membrane structure and aβ peptide interactions545766/fulltext01.pdf · oxidized...
TRANSCRIPT
Oxidized Phospholipids:
Roles in Lipid Membrane Structure and Membrane ‐ Aβ Peptide Interactions
Nguyen Vu Khanh Linh
Degree Thesis in Chemistry, 30 ECTS
Master’s level
Report passed: 8th February 2011
Supervisor: Gerhard Gröbner
Examiner: Magnus Wolf‐Watz
Table of Contents Abstract ................................................................................................................................................... 2
Background.......................................................................................................................................... 3
Biological Membranes..................................................................................................................... 3
Alzheimer’s Disease......................................................................................................................... 4
Macromolecular Crowding.............................................................................................................. 5
Lipid Classes..................................................................................................................................... 5
Aim of the Project................................................................................................................................ 6
Biophysical Techniques for Studying Lipid Membranes...................................................................... 6
Differential Scanning Calorimetry (DSC) ......................................................................................... 6
Solid State NMR............................................................................................................................... 8
Material and Methods......................................................................................................................... 9
Material ........................................................................................................................................... 9
Sample Preparation for DSC Studies ............................................................................................... 9
Sample Preparation for NMR Studies ........................................................................................... 10
DSC Measurements ....................................................................................................................... 10
Solid State NMR Measurements ................................................................................................... 10
Results ............................................................................................................................................... 11
Differential Scanning Calorimetry (DSC) ....................................................................................... 11
NMR Studies .................................................................................................................................. 21
Discussion.......................................................................................................................................... 29
Effect of Oxidized Phospholipids on DMPC Membranes Studied by DSC ..................................... 29
Crowding Effect ............................................................................................................................. 30
Effect of Oxidized Phospholipids on DMPC Membranes Studied by Solid State NMR ................. 31
Difference between H2O and D2O as Solvents for DMPC/OxLis in NMR Experiment ................... 33
Interplay between Aβ Peptide and Oxidized Lipids Containing Membranes................................ 33
Conclusion ......................................................................................................................................... 34
Acknowledgement................................................................................................................................. 35
References............................................................................................................................................. 36
1
Abstract Lipid membranes play a crucial role in cellular organization and function. Since membrane compositions can change during aging and due to pathological alterations, numerous researches focus on the elucidation of the role of membranes in amyloidogenic diseases,
most prominently Alzheimer’s disease (AD). AD is caused by the aggregation of amyloid‐β peptide, a process which can accelerate in the presence of membrane interfaces. Since oxidized phospholipids are considered to be involved in pathological conditions like AD, the main aim of this project is to identify the impact of oxidized lipids on the structural and dynamical organization of biological lipid model membranes and occurring membane‐peptide interactions. For this purpose two biophysical methods were applied: differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR). The model membranes used, were composed of neutral phosphatidylcholine lipids (DMPC) containing varying amounts of oxidized lipid species (PazePC, PGPC). The obtained results reveal a major impact of oxidized lipids on the organization of DMPC bilayer. However, the size of the effect depends on the pH present and the concentration of oxidized lipid present in the membrane system. Moreover, the oxidized lipid‐containing membrane inhibits structural changes upon incorporation of amyloid peptide by either dynamic or orientation changing.
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Background
Biological Membranes Biological membranes play a central role in both the structure and function of all cells, prokaryotic and eukaryotic organism [1]. Lipids, proteins, and carbohydrates are the main components of biological membranes. Our focus in this study is on the membrane matrix forming lipid molecules (Figure 1). Membrane lipids form a two‐dimensional bilayer, in which membrane proteins are embedded to fulfill their function. In general, this bilayer has to be in a fluid‐like, liquid‐crystalline state for the membrane to fulfill its biological tasks. This fluidity is modulated by the motional dynamics of the lipid molecules and especially their hydrophobic fatty acid chains; and regulation in the fluidity strongly influences the permeability and mechanical properties of the membrane, as well as the activity of membrane proteins [2].
Figure 1: General membrane structure (adapted from Gerhard Gröbner, Umeå University, Sweden).
The most commonly found membrane lipids are glycerophospholipids, which are derivatives of sn‐glycero‐3‐phosphoric acid. Glycerophospholipids have the phosphate at the sn‐3 position of glycerol, and two long‐chain hydrocarbons attached to sn‐1 and sn‐2 through ester or ether linkages [1]. Glycerophospholipids containing polyunsaturated fatty acids are highly prone to oxidative modifications under various conditions including various phases of disease progression to form oxidized phospholipids (oxPLs) [3]. Lipid oxidation in vivo may involve both enzyme‐catalyzed and non‐enzymatic reactions [4]. Since the polarity and shape of phospholipids oxidation products may differ significantly from the structure of their parent molecules, they are supposed to change the properties of biological membranes. Moreover, they can alter lipid‐lipid and lipid‐protein interactions as well as membrane protein functions [4].
The main focus of this project is to provide a general molecular understanding of the interplay between oxidized lipids and their membrane environment and any occurring consequences for interactions between these membranes and amyloid‐β peptide, the key component in AD disorder.
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Alzheimer’s Disease Alzheimer’s disease is the predominant form of senile dementia and is characterized by neuritic plaques and cerebrovascular amyloid deposits [5]. Amyloid‐β peptide (Aβ) is the main compound of Alzheimer’s amyloid deposits, which is a cleavage from the amyloid precursor protein (APP). The most common variant, Aβ1‐42 composes of 28 extracellular (hydrophilic) and 14 transmembrane (hydrophobic) residues; while the shorter species, Aβ1‐40 has only 12 residues in transmembrane domain together with 28 extracellular residues. The presence of both hydrophilic and hydrophobic residues results in an amphiphatic behavior of the peptide. At higher concentrations under suitable conditions of pH and salt concentration, Aβ undergoes conformational changes from a non‐toxic random‐coil structure into aggregation prone β‐sheet‐like conformation, which further progress to oligomeric and finally fibrillar structures, a process shown to be toxic to neurons [5]. Due to its amphiphatic property, Aβ peptide can exhibit electrostatic interactions with other components which exist intra‐ and extracellular cell; and there is evidence that Aβ fibril assembly is dependent on interactions with biological surfaces, such as membranes [6]. Figure 2 shows the aggregation process of the peptide at the membrane surface. Since oxidized phospholipids once formed can alter the membrane properties, it is important to study the interaction between Aβ peptide and membrane surface in the presence of oxidized phospholipids species. Nevertheless, the peptide originates originally from the membrane where it is in a transmembrane position; a situation which might it even protects it from release or forces it already into ion‐channel oligomeric structures at this location [7]. However, there is no knowledge about the role of oxidized lipids in these processes.
Figure 2: Schematic model for Aβ peptide interactions with membrane. Top: Electrostatic adsorption to membrane surfaces. Bottom: Insertion of Aβ peptide into membranes (adapted from Bokvist, Gröbner, J. Mol. Biol. (2004) 335, 1039‐1049 [7]).
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Macromolecular Crowding
In general, membranous system found inside a cell experiences an effect, called “Macromolecular Crowding”, which describes a restricted compartment congested by macromolecules. Therefore there exist only a reduced free volume for biomolecules; an effect which favors compact protein states e.g. by reduced protein solubility; a process which causes increased self‐association of amyloidogenic proteins [8]. However, there are no biophysical studies to investigate the impact of crowding on the phase behavior of cellular membrane matrices directly. Therefore we investigated here any eventual effect of crowding agents on the physico‐chemical behavior of specific lipid model membranes.
Lipid Classes
Since lipid membrane contains about thousand different pieces of phospholipids, well defined biological model membranes have to be used to study structure‐function relationship of a lipid component in cell membranes [9]. In this work, the main phospholipids species used here is DMPC (1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine), which has two carbons of a glycerol backbone connected to 14‐carbon long fatty acid chains while the other carbon is connected to phosphocholine group (Figure 3). To study the impact of oxidized lipids on the properties of DMPC lipid membranes, two different oxidized lipids were incorporated in varying amounts, namely PazePC (1‐palmitoyl‐2‐azelaoyl‐sn‐glycero‐3‐phosphocholine) and PGPC (1‐palmitoyl‐2‐glutaryl‐sn‐glycero‐3‐phospho‐choline) which have carboxylic functional groups at sn‐2 position of glycerol, respectively.
DMPC
PazePC
PGPC
Figure 3: Structures of lipids used in this project: DMPC (1, 2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine), PazePC (palmitoyl‐2‐azelaoyl‐sn‐glycero‐3‐phosphocholine), PGPC (1‐palmitoyl‐2‐glutaryl‐sn‐glycero‐3‐phosphocholine).
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Membrane lipids display different polymorphism, depending not only on the structure of the lipid molecule itself and on its degree of hydration, but also on temperature, pressure, ionic strength, and pH [10]. Therefore, in this project, the impact of varying pH and of oxidized lipids amounts on the lipid membrane behavior was also investigated with the help of various biophysical techniques.
Aim of the Project
The first aim of this project is the characterization of the structural and dynamic organization of defined DMPC lipid bilayer in the presence of different OxLis. The impact on membrane fatty acid order and dynamics in the membrane hydrophobic core, and occurring long range effects on the polar membrane region. Defined OxLis such as PazePC and PGPC were used in this study. OxLis concentration and pH of the environment were varied to observe their effect on membrane structure. Solvent effect was studied by comparison between normal water and heavy water (D2O) as solvents for NMR experiments. Crowding effect on OxLis is also investigated here by incorporation of Ficoll to the lipid dispersions.
Secondly, the impact of OxLis species on the interaction between Aβ peptide and membrane matrix is investigated. In order to answer for the question if oxPLs may affect the membrane structure upon electrostatic interaction with the peptide, NMR approach is applied to get molecular insight into the system.
Biophysical Techniques for Studying Lipid Membranes
Differential Scanning Calorimetry (DSC)
DSC is the most widely used of all the thermal analysis techniques to obtain information on thermal changes in a membrane sample and therefore its phase behavior and changes induced by specific lipid constituents. In general, the device has two chambers (Figure 4A), with one for the sample and one for the reference (often buffer used). And the signal from the instrument depends on the difference in energy used to bring both chambers up to the same temperature upon heating [1]. When the sample undergoes a thermo‐tropic phase transition, the sample and the reference behave differently, since the sample needs a very high amount of energy to undergo the phase transition. As a result, change in heat flow into the sample and the reference cells occurs, which can be recorded. The output of the instrument is a plot of differential heat flow (dE/dt) as a function of temperature in which the intensity of the signal is directly proportional to the scanning rate (dT/dt) [10].
For the gel to liquid‐crystalline phase transition of simple phospholipids, important information can be achieved by analyzing these thermograms (Figure 4C). Tm is the highest temperature during the transition. T1/2 is the width of the peak at half high, which determines the sharpness of asymmetric peaks. Enthalpy of transition can be integrated from the peaks.
ΔHcal = ∫ TpdC
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DSC technique has been of primary importance in studies of lipid phase transition in model biological membranes [9]. In order to have a precise measurement, the reference should have exactly the same composition with the sample cell, but without the target lipids.
A B
C
D
Figure 4: Concept of DSC studies: A; Principle of DSC measurement, B: a DSC machine, C: DSC thermogram of a phospholipid, D: Scheme of phase transitions between gel states (Lβ’), ripple state (Pβ’), and liquid‐crystalline phase (Lα’); (adapted from C. Aisenbrey).
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Solid State NMR
NMR has proved to be a very powerful tool in the study of lipid polymorphism, especially 31P and 14N solid state NMR [10]. Each phospholipids molecule has at least one phosphor nucleus in its head group, and the same is true with the nitrogen nucleus residing in the choline head group segments of phosphocholine lipid species. The main methods used in this work for study transition between various lipid phase structures were 31P and 14N solid state NMR.
31P NMR exhibits 6.6% of the sensitivity of 1H NMR, and 31P is one of the most sensitive nuclei for the NMR experiment [11]. 31P NMR senses the dynamic head group region via the P nucleus, whose behavior therefore reflects the membrane surface properties. The 31P line shapes (Figure 5, right) are a result of various dynamic averaging processes and can normally be analyzed based on the knowledge of the particular lipid head group structure and the existing dynamic processes [10]. However, they are not theoretically in a one‐to‐one correspondence with the particular phase structure [11]. In general, in mixed phospholipids bilayers the static 31P NMR spectra are the result of overlapping sub‐spectra of the individual lipid species and therefore difficult to analyze. Therefore, in high resolution solid state NMR, the sample is spun at the magic angle to achieve isotropic NMR peaks whose positions indicate specific lipid species. This method is called magic angle spinning (MAS), with the angle 54.70 according to the scaling factor [11]:
(3cos2θ ‐ 1)
where θ is the angle between the axis of rotation and the external magnetic field. When θ = 54.70
this term is 0 and under fast spinning conditions the anisotropic chemical shift contributions to the NMR spectrum disappear and only sharp resonances remain, whose position presents the isotropic chemical shifts, whose values are specific for different lipids. Figure 5 shows examples of static 31P and 14N NMR spectra and upon MAS conditions.
The nitrogen nucleus in lipid head group can also be useful for NMR experiment by it dominant population. With nuclear spin of I>1, 14N has an electric quadrupolar moment which arises from an asymmetrical distribution of the nuclear charge [12]. The quadrupolar moment interacts with the electric field by quadrupolar interaction, which can affect the Zeeman interaction. The quadrupolar interaction is canceled out in the solution, but not in solid state sample; hence, it results in the Pake pattern as can be seen in Figure 5 (left).
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31P NMR 14N NMR
Figure 5: Scheme of 31P and 14N Solid State NMR with and without spinning on DMPC/DMPG and DMPC/DDAB vesicles (adapted from [13]).
Material and Methods
Material 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC), 1‐palmitoyl‐2‐azelaoyl‐sn‐glycero‐3‐phospho‐choline (PazePC), 1‐palmitoyl‐2‐glutaryl‐sn‐glycero‐3‐phosphocholine (PGPC), were from Avanti Polar Lipids (Alabaster, USA). Three different buffers were used in this work: buffer A (pH 5.0, 20 mM CH3COONa, 10 mM KCl, 140 mM NaCl, 0,5 mM EDTA), buffer B (pH 7.4, 10 mM Tris, 10 mM KCl, 140 mM NaCl, 0,5 mM EDTA), and buffer C (pH 9.5, 20 mM glycine, 10 mM KCl, 140 mM NaCl, 0,5 mM EDTA).
Sample Preparation for DSC Studies Appropriate amounts of the various lipids used were dissolved in chloroform: methanol (2:1 v/v). The solvent was removed by rotary evaporation and the lipid residue was hydrated. The solution of lipid in water was then further dried overnight under vacuum condition to remove all organic solvents. The dried lipid powder was collected and stored in the freeze. For each experiment, few milligrams of lipid powder were hydrated by suitable buffer and treated with several freeze‐thaw cycles to obtain homogenous mixtures of lipid. The liposomes were used directly in DSC measurements after the preparation.
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Sample Preparation for NMR Studies The solid lipid mixtures was dissolved in appropriate buffer at 1:60 molar ratios (lipid/water) and homogenized by several freeze‐thaw cycles. The liposome pellet was packed into a 4 mm MAS NMR rotor for NMR studies.
DSC Measurements The measurements were performed on a VP‐DSC calorimeter (MicroCal, Inc., Northamton, MA, USA). All the samples were degassed before each run. A volume of 0.5 ml of 3mM liposomes was injected into the sample cell, and the same volume of appropriate buffer was placed into the reference cell. The cells were at first heated from 5°C to 45°C (scanning rate 60°C/h), then cooled down back to 5°C with the same scanning rate. Then a scan from 5 °C to 45 °C with scanning rate 2 °C/h was preformed and the results of these scans are presented.
Solid State NMR Measurements 31P measurements were carried out on a 400 MHz Infinity spectrometer (Chemagnetics, U.S.A) with a 4‐mm double resonance MAS probe. The isotropic shift of DMPC vesicles was used as an external reference. The static experiments were acquired by applying a Hahn echo sequence with an inter‐pulse delay of 50 µs. 31P MAS NMR measurements were done under proton decoupling using a single π/2 pulse with 5.5 µs pulse length.
14N MAS NMR spectra were acquired using a rotor‐synchronized quadrupole echo sequence with π/2 pulse duration of 7.3 µs. For static 14N NMR spectra a 200‐µs interpulse delay was utilized. The isotropic shift of DMPC vesicles were used as an external reference (0.0 ppm).
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Results
Differential Scanning Calorimetry (DSC)
Thermotropic phase behavior of pure DMPC dispersions
Figure 6: DSC heating thermogram of 3 mM DMPC dispersions in buffer B (pH 7.4).
The DSC heating scan of a fully hydrated aqueous dispersion of DMPC exhibits two endothermic transitions (Figure 6). The first event at 13.00C shows the transition from a planar gel (Lβ’) to the ripple gel (Pβ’). The second event at 23.1
0C corresponds to the transition from a Pβ’ to a lamellar liquid‐crystalline phase (Lα).
Effect of OxLis on DMPC Membranes
Lipid vesicles made of DMPC lipids with varying concentrations of different OxLis were examined under the same DSC conditions as for pure DMPC bilayers, namely from 50C to 450C with a 20C/h scan rate.
Clearly the phase behavior of DMPC bilayers is severely changed upon the presence of PazePC. The main transition observed for all three different concentrations of PazePC, seems to be a supercomposition of two peaks. As the PazePC concentration increases under the same pH condition (pH7.4), one can see that the sharp component is shifted to lower temperatures while the broad
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component stays at the same position (Figure 7). The total enthalpy (Table 1) of both components increases according to an increasing PazePC concentration.
Figure 7: DSC heating thermogram of DMPC in the present of: 2% (black), 5% (blue) and 10% (red) PazePC. The lipid concentration is 3 mM in buffer B (10 mM Tris, pH 7.4).
DMPC/10%PazePC at Different pH
In the presence of 10% PazePC there is a large impact on the DMPC transition as a function of the pH (Figure 8). The pre‐transition disappears at all three pH conditions (5.0, 7.4 and 9.5). At pH 5.0 and 7.4, the main transition behavior looks quite similar. Here, the thermograms seems to be asymmetric consisting of two overlapping thermal events; one of these components is considerably sharper than the other. The sharp component is shifted to lower temperatures compared to a pure DMPC main transition while the broad component is shifted to a slightly higher temperature. The total enthalpy of two components is larger than in pure DMPC (Table 1). There is a reducing of total enthalpy when the environment is more basic. Especially, at pH 9.5, the main transition is almost abolished.
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Figure 8: DSC heating thermogram of DMPC in the present of 10% PazePC at three different buffers: A (pH 5.0), B (pH 7.4) and C (pH 9.5).
Crowding Effect
To study the effect of the crowding environment on the phase behavior of lipids membranes, the lipid systems were also studied by DSC in the presence of 200 mg/ml Ficoll (see Figure 9, 10, 11, 12). It can be seen that even DMPC alone is also affected by the crowding conditions. The significant effect caused by crowding is a reduction in enthalpy of the gel to liquid‐crystalline transition. Results are summarized in Table 1 and 2.
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0
2
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6
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DMPC with Ficoll DMPC without Ficoll
Cp
(kca
l/mol
e/o C
)
Temperature (oC)
Figure 9: DSC heating thermogram of pure DMPC in buffer B (pH 7.4) with (red) and without (black) 200mg/ml Ficoll.
10 15 20 25 30 35 40
0
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4
6
8
10
12 paze10% pH7.4 with Ficoll paze10% pH7.4
Cp
(kca
l/mol
e/o C
)
Temperature (oC)
Figure 10: DSC heating thermogram of DMPC/10% PazePC in buffer B (pH 7.4); the red line corresponds to a lipid mixture with Ficoll present.
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paze10% pH5.0 with Ficoll paze10% pH5.0
Cp
(kca
l/mol
e/o C
)
Temperature (oC)
Figure 11: DSC heating thermogram of DMPC/10% PazePC in the presence of buffer A (pH 5.0). The red line corresponds to a lipid mixture with Ficoll present.
10 15 20 25 30 35 40-0,5
0,0
0,5
1,0
1,5
2,0
paze10% pH9.5 with Ficoll paze 10% pH9.5
Cp
(kca
l/mol
e/o C
)
Temperature (oC)
Figure 12: DSC heating thermogram of DMPC/10% PazePC in the presence of buffer C (pH 9.5; black line) containing 200mg/ml Ficoll (red line).
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Table 1: Phase transition temperature, enthalpy for gel‐liquid crystalline transition of pure DMPC in buffer B (pH 7.4) with and without Ficoll.
pH 7.4 DMPC
Without Ficoll With Ficoll
Tm (⁰C) 23.0 22.9
∆H (kcal/mol) 5.38 5.21
Table 2: Phase transition temperature, enthalpy for gel‐liquid crystalline transition of DMPC/10%PazePC with and without Ficoll. Tm1 and Tm2 are temperature for the sharp component and the broad component, respectively. At pH 9.5, these transition peaks were too broad to be integrated.
pH 5.0 pH 7.4 pH 9.5 DMPC/10%PazePC Without Ficoll
With Ficoll Without Ficoll
With Ficoll Without Ficoll
With Ficoll
Tm1 (⁰C) 21.3 21.5 21.2 21.6 ‐ ‐
Tm2 (⁰C) 23.6 23.5 23.9 23.6
∆H total (kcal/mol)
7.00 5.84 6.80 6.55 ‐ ‐
DMPC/2%PazePC at Different pH
At lower concentrations of PazePC, namely 2%, the effect on DMPC bilayers is much less pronounced than the one observed for 10%PazePC. The pre‐transition can be seen as 7.6 0C, but it is much broader than in pure DMPC and the intensity is reduced. At pH 7.4 and 9.5 a second component around the main transition area can be seen (Figure 13). The enthalpy is highest at pH 7.4, and quite similar at the other two pH conditions. Results are shown in Table 3.
16
20 25
0
10
20
paze2% pH9.5 paze2% pH5.0 paze2% pH7.4
Cp
(kca
l/mol
e/o C
)
Temperature (oC)
Figure 13: DSC heating thermogram of DMPC in the present of 2% PazePC at three different buffers: A (pH 5.0), B (pH 7.4) and C (pH 9.5).
Table 3: Phase transition temperature, enthalpy for gel‐liquid crystalline transition of DMPC/2%PazePC with and without Ficoll. For crowding effect, the thermograms are not shown.
pH 5.0 pH 7.4 pH 9.5 DMPC/2%PazePC Without Ficoll
With Ficoll Without Ficoll
With Ficoll Without Ficoll
With Ficoll
Tm (⁰C) 22.9 22.9 22.9 23.0 22.8 22.9
∆H (kcal/mol) 5.97 5.19 6.58 6.14 5.94 5.55
DMPC/2%PGPC at Different pH
Similar to PazePC, PGPC has a carboxylic group at the end of sn‐2 chain, but it has 4C less than in the sn‐2 chain. At 2% PGPC concentration, the pre‐transition can still be observed at around 80C, and the main transition is almost the same as in pure DMPC (Figure 14). The peaks were broadened compared to pure DMPC. At pH 7.4 and 9.5, there is another component appearing in the main transition. The total enthalpy increases according to increasing pH. The crowding effect also reduces the enthalpy of the transition peaks, with values given on Table 4.
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20
0
5
10
15
PC/PGPC 2% pH9.5 PC/PGPC 2% pH7.4 PC/PGPC 2% pH5.0
Cp
(kca
l/mol
e/o C
)
Temperature (oC)
Figure 14: DSC heating thermogram of DMPC in the presence of 2% PGPC at three different buffers: A (pH 5.0), B (pH 7.4) and C (pH 9.5).
Table 4: Phase transition temperature, enthalpy for gel‐liquid crystalline transition of DMPC/2%PGPC with and without Ficoll.
pH 5.0 pH 7.4 pH 9.5 DMPC/2% PGPC Without Ficoll
With Ficoll Without Ficoll
With Ficoll Without Ficoll
With Ficoll
Tm (⁰C) 22.9 23.0 22.8 23.0 22.8 22.9
∆H (kcal/mol) 5.84 5.73 5.96 4.43 6.49 5.56
Effect of Aβ peptide Incorporated into DMPC/PazePC Dispersions
Upon incorporation of Aβ peptide, the transition behavior of DMPC/10%PazePC has changed with respect to transition temperature and enthalpy. As can be seen in Figure 15 the sharp component in peptide‐containing lipid dispersions was broadened and shifted to lower temperatures, while the broad component was slightly shifted towards higher temperatures. Table 5 shows temperature and enthalpy values of the lipid mixture prior and upon incorporation of the peptide. Enthalpy of the gel‐liquid crystalline transition was significantly reduced upon the presence of peptide.
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Figure 15: DSC heating thermogram of DMPC/10%PazePC (black) in buffer B (pH 7.4) and upon incorporation of Aβ peptide (red) at a molar ratio (DMPC/10%PazePC)/peptide 100/1.
Crowding Effect on DMPC/PazePC/Peptide Dispersions
Figure 16 shows three thermograms of peptide free DMPC/10%Paze vesicles (black), DMPC/10%PazePC vesicles containing Aβ peptide at a 100/1 molar ratio without (red) and with ficoll crowding agent (200 mg/ml Ficoll; blue line). It can be seen that in the presence of Ficoll the sharp component in the thermogram is shifted to higher temperature. The total enthalpy of two components also increases.
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Figure 16: DSC heating thermogram of DMPC/10%PazePC in buffer B (pH 7.4) and upon incorporation of Aβ peptide (red) at a molar ratio (DMPC/10%PazePC)/peptide at 100/1 molar ration prior und upon the presence of Ficoll. Black line: DMPC/10%PazePC, red line: DMPC/10%PazePC/peptide, blue line: DMPC/10%PazePC/peptide + Ficoll 200mg/ml.
Table 5: Phase transition temperature, enthalpy for gel‐liquid crystalline transition of DMPC/10%PazePC/peptide with and without Ficoll. Tm1 and Tm2 are temperature for the sharp component and the broad component, respectively. Buffer used was buffer B, pH 7.4.
DMPC/10%PazePC (DMPC/10%PazePC)/peptide 100/1 molar ratio
(DMPC/10%PazePC)/peptide 100/1 molar ratio with 200
mg/ml Ficoll
Tm1 (⁰C) 21.2 20.7 21.3
Tm2 (⁰C) 23.9 24.3 23.8
∆H (kcal/mol) 6.80 5.05 6.59
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NMR Studies
Behavior of DMPC Bilayers Studied by 31P Solid State NMR
A typical NMR spectrum, also called “powder pattern” of multilammellar DMPC vesicles can be seen for different temperatures in Figure 17A. Upon increase in temperature the chemical shift anisotropy (the width) of the NMR powder pattern becomes smaller. Figure 20 shows the temperature dependence of the relative chemical shift values for the high‐field edge of the NMR spectra. There is a decrease in chemical shift values at around 286K, where the pre‐transition occurs. After that, the chemical shift increases back to the same level as before, and decreases again from 295K, where the main phase transition occurs. .
Effect of Oxidized Phospholipids on DMPC Studied by 31P Solid State NMR
DMPC/10%PazePC at Different pH
At pH 7.4, in comparison with pure DMPC dispersions, the NMR spectra of mixed DMPC/PazePC bilayers (10 %mole PazePC) are similar, but at a specific temperature range two overlapping powder sub‐spectra can be observed, named inner domain and outer domain (Figure 17). Compared to the relevant DSC diagram, the second high‐field NMR peak appears in the temperature region, where the second broad phase transition can be seen (from 295K to 299K). With increasing temperature these inner NMR peaks move closer to the peak of the wider sub‐spectra. For the main outer peak, the chemical shifts have the highest values while the small peaks appear, and are in general higher in the liquid crystalline phase (above phase transition). Figure 18 shows the correspondence between DSC scan and NMR measurement spectra of the sample. In Figure 21 the temperature dependence of the chemical shift values of the high‐field peaks for both sub‐spectra are plotted.
40 20 0 -20 -40
ppm40 20 0 -20 -40
ppm
BA
0C
15
18
22
25
35
Figure 17: 31P NMR spectra at 400MHz proton frequency of multilamellar DMPC (A) and DMPC/10%PazePC (B) vesicles in H2O in buffer B (pH 7.4).
21
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ppm20 30
0
5
10
Cp
(kca
l/mol
e/o C
)
Temperature (oC)
B A
15
21
22
23
24
35
0C
Figure 18: Phase behavior of DMPC/10%PazePC at pH7.4 studied by DSC (A) and 31P NMR (B).
DMPC/2%PazePC and DMPC/5%PazePC
Under the same pH condition, DMPC bilayers containing PazePC at a 2 mole % and 5 mole%, generate in NMR experiments complex spectra, reflecting a polymorphic membrane behavior. However, the temperature dependent changes in the chemical shift values of the up‐field edge in the powder pattern (Figure 22) suggest a change in motional reorientation of the lipid. At higher PazePC concentrations, the width of the NMR spectra is reduced, indicating an increase in motional freedom in the membrane interface area.
Figure 19 shows the NMR spectra of DMPC containing 2 mole% PazePC at pH 7.4. Since the start of the theses measurements, additional NMR resonances appeared compared to the spectra obtained on DMPC/10%PazePC vesicles. Over time the intensities of these components increased. Above 270C, the sample started to behave differently, resulting in complex spectra. After the end of the static NMR experiments, the sample was studied by MAS NMR again, and more than one isotropic peak was observed (data not shown), indicating lipid fragmentation products.
At pH 9.5 the lipid mixture seems to be unstable and therefore a normal wideline NMR powder pattern was observed only in the first hours of the NMR studies; later this sample also degraded rapidly (data not shown).
22
A B
20 0 -20 -40
0C
21 22 23 24 25 26
0
5
10
15
20
ppm
Cp
(kca
l/mol
e/o C
)
Temperature (oC)
Figure 19: Phase behavior of DMPC/2%PazePC at pH7.4 studied by DSC (A) and 31P NMR (B).
Figure 20: The temperature dependence of chemical shifts (high‐field edge) in 31P NMR powder pattern of DMPC multilammellar dispersions
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26
23
Figure 21: NMR chemical shifts (high‐field edge) of DMPC/10%PazePC vesicles as a function of temperature.
Figure 22: Chemical shifts versus temperature for DMPC/PazePC at different PazePC concentrations.
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Difference between H2O and D2O as Solvent for DMPC/OxLipids in NMR Experiments
Since D2O is an essential solvent in NMR experiments, but has slightly different properties compared to H2O a comparison of the behavior of DMPC/Paze10% vesicles in buffers composed of H2O versus D2O was carried out. There are clearly differences in the observed NMR powder patterns of DMPC/10%PazePC in these two environments. In D2O the splitting occurs at lower temperatures compared to samples in the presence of H2O (Figure 23).
40 20 0 -20 -40
ppm40 20 0 -20 -40
ppm
0CA B
15
21
22
23
24
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Figure 23: 31P NMR spectra at 400MHz proton frequency of multilamellar DMPC/10%PazePC vesicles in H2O (A) and D2O (B). Buffers pH 7.4, 10 mM Tris, 10 mM KCl, 140 mM NaCl, 0,5 mM EDTA in H2O/ D2O.
DMPC/2%PGPC
At 2 mole % PGPC concentration, the NMR powder pattern of these mixed DMPC/PGPC vesicles has a similar behavior as observed for pure DMPC bilayers (Figure 24). The only effect is a decrease in the size of the chemical shift anisotropy of the powder pattern, as can be seen in Figure 25. The less negative chemical shift values for the high‐field edge of the NMR spectra compared to spectra of pure DMPC bilayers indicate a less ordered lipid structure.
25
40 20 0 -20 -40
ppm
0C
35
15
22 25
18
Figure 24: 31P NMR spectra at 400MHz proton frequency of multilamellar DMPC/2%PGPC vesicles in buffer B containing H2O.
Figure 25: Chemical shifts versus temperature for DMPC and DMPC/2%PGPC in buffer B (pH 7.4). The blue solid line represents for DMPC/2%PGPC, and the green one represents for pure DMPC dispersions.
Isotropic Peaks in 31P MAS NMR
The 31P MAS NMR isotropic peak of pure DMPC bilayers at a spinning speed 6 kHz appears at ‐0.9 ppm at 308K, as can be seen in Figure 26. In the presence of certain amounts of PazePC under different pH conditions, the chemical shifts vary slightly. At the presence of 10 mole % of PazePC, the isotropic peak of 31P is significantly affected at high pH conditions (pH 9.5). The same behavior is observed for 2 mole % PazePC containing DMPC membranes under neutral and basic conditions. Moreover, at 2mole %PazePC there was more than one single isotropic peak to be observed in the spectra, indicating that the sample was unstable. Values of all the isotropic peaks are summarized in Table 6.
26
80 60 40 20 0 -20 -40 -60 -80
ppm
31P MAS DMPC/10azePC pH 7.4
Figure 26: 31P MAS spectra at 6 kHz spinning speed of DMPC/10%PazePC at 308K, buffer B (pH 7.4). The isotropic peak is at ‐1.0 ppm.
Table 6: Isotropic peaks for DMPC and different mixtures with oxidized lipids. σ3 is the main isotropic peaks, whereas
σ1 and σ2 are small peaks developing during the experiments with low intensities.
σ1 (ppm) σ2 (ppm) σ3 (ppm)
DMPC -0.9
DMPC/10%PazePC pH7.4 -1.0
DMPC/10%PazePC pH9.5 -1.5
DMPC/2%PazePC pH7.4 -0.27 -0.82 -1.3
DMPC/2%PazePC pH9.5 -0.26 -0.8 -1.3
DMPC/2%PGPC pH7.4 -0.97
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Effect of Aβ Peptide on DMPC/PazePC Dispersions
Both 14N and 31P solid state NMR experiments were carried out on DMPC/10%PazePC vesicles prior and upon incorporation of Aβ peptide (Figure 27 and 28).
In 31P NMR spectra, it can be seen that at high temperature (350C) there is no significant difference in DMPC/10%azePC behavior when the peptide is present (Figure 27). At gel‐to‐liquid crystalline transition temperature (230C), however, there is reducing of powder pattern width, and the high field edge in the powder pattern was shifted to less negative chemical shift. Two splitting indicating two phases in the powder pattern can still be seen upon addition of peptide.
For 14N NMR spectra of DMPC/10%PazePC upon addition of peptide, the splitting in the powder pattern decreases about 0.5 kHz at both room temperature and higher temperature (Figure 28). At 230C, moreover, there are other components in the spectra compare to DMPC/10%PazePC only.
230C 350C
Figure 27: 31P NMR spectra at 400MHz proton frequency of multilammellar DMPC/10%PazePC vesicles at 350C and 230C, respectively. The upper panel shows spectra of peptide‐free vesicles and the lower panel spectra obtained of vesicles upon peptide incorporation at a 100/1 lipid to peptide molar ratio.
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350C 230C
Figure 28: 14N NMR spectra at 400MHz proton frequency of multilammellar DMPC/10%PazePC vesicles at 350C and 230C, respectively. The upper panel shows spectra of peptide‐free vesicles and the lower panel spectra obtained of vesicles upon peptide incorporation at a 100/1 lipid to peptide molar ratio.
Discussion
Effect of oxidized Phospholipids on DMPC Membranes Studied by DSC
As seen in our studies, lipidperoxidation can cause disordering of lipid membranes by incorporation of oxidized lipid species; a process which can alter the polarity of the bilayer interior, the chain‐chain hydrophobic interactions and chain‐water interactions [14].
DSC results in this work indicate that the presence of oxidized phospholipids has significant effects on the physico‐chemical behavior of DMPC membranes, especially at high contents and/or a basic environment.
As seen in Figure 7 the presence of PazePC lipids induces the formation of two components in the DSC thermogram of mixed DMPC/PazePC membranes, with the second component already appearing at 2 mole% PazePC. However, at 2 mole% and 5 mole% PazePC, the second component is present but not totally separated from the sharper component. In general, an increasing PazePC concentration shifts the transition temperature to lower values; a behavior which indicates a
29
perturbation of DMPC’s gel phase by the presence of oxidized lipids. This behavior can be seen for many membrane effectors molecules including e.g. peptides such as gramicidin as shown previously [15].
In the presence of 10% of PazePC, two thermal events, visible as a sharp and a broad component, were clearly observed in the gel‐to‐liquid‐crystallize transition area of the DSC profile (Figure 8). The sharp and broad components correspond to differential melting of OxLi‐poor and OxLi‐rich DMPC regions, respectively; an observation also seen upon incorporation of cholesterol [16]. At pH 7.4 and 5.0, the transition of the sharp component was shifted to lower temperatures, indicating a destabilization of the gel state in OxLi‐poor domain. The broad component, on the other hand, shifted to slightly higher temperatures; a behavior indicating a destabilization of the liquid‐crystallize phase in OxLi‐rich DMPC lipid domains. The total enthalpy of the two components in PazePC‐containing DMPC bilayer is larger than in pure DMPC dispersions, but changes according to the pH of the environment. Under acidic condition (pH 5.0) the total enthalpy is higher than under neutral conditions (pH 7.4), and at high pH conditions (pH 9.5), the transition is almost abandoned. For DMPC vesicles containing 2 mole % PazePC, the measured transition temperature was similar to the one observed for pure DMPC, but the widths of the transition peak were affected by the pH conditions present (Figure 13). At pH 5.0, there is only a single peak indicating the gel‐to‐liquid‐crystalline transition. This suggests that under these acidic environment, the presence of 2% PazePC does not induce another component in the DMPC membrane due to the fully protonated, uncharged carboxyl group at the sn‐2 chain. The second component starts to appear at pH 7.4 and 9.5, and Figure 6 shows that the transition peaks are broaden when the pH is higher. When studying DMPC bilayers with PGPC lipids incorporated at concentrations of 2 mole%, nearly the same effect is induces as in the presence of 2 mole% PazePC (Figure 14). The sn‐2 chain in PGPC has 4‐carbons less than PazePC; this difference in structure does not cause significant differences in bilayer behavior at this concentration. At pH 5.0 there is a single peak; an observation indicating that the carboxyl group in PGPC is still uncharged. However, the transitions seem to be composed of two overlapped peaks at higher pH. That maybe due to the partially deprotonated and totally deprotonated of carboxyl groups at pH 7.4 and 9.5, respectively.
Crowding Effect The crowding effects were almost the same for all mixtures in the study (DMPC/10%PazePC, DMPC/2%PazePC, DMPC/2%PGPC). The most pronounced effect of the presence of crowding agent (200mg/ml Ficoll) can be seen in a reduction of the enthalpy for the gel‐to‐liquid crystalline phase transition; and variously a reduction in the width of the phase transition. However, the phase transition temperature of the sharp component increases, indicating a more stable gel state. That implies the crowding environment favors the more ordered membrane structure; in the other words, the gel state is stabilized. The effect is similar when the Aβ peptide was incorporated to the DMPC/10%PazePC dispersions using buffer B which contained 200 mg/ml Ficoll. In OxLis‐poor domain Ficoll induces more stable gel state by higher phase transition temperature, while the OxLis‐rich domain was not significantly affected.
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Effect of Oxidized Phospholipids on DMPC Membranes Studied by Solid State NMR
Since lipid bilayers are spontaneously formed by phospholipids molecules after they are hydrated, it is necessary to study the structure and properties of these lipid bilayers composed of well defined molecular species in the presence of defined excess of water [9]. Here 1:60 molar ratio of lipid to water was used for the NMR experiments.
The lipid bilayer is highly anisotropic, and the main motion of phospholipids is an axial rotation about the lipid long axis in the bilayer [11]. This rotational process and additional wobbling of the lipid long axis and internal motion result in a powder pattern like NMR line shapes as can be seen in Figure 17A. The observed line shape is composed of the sum of all orientations present in powder dispersion [13], where different molecular orientations present in the sample give rise to different spectra frequencies. These lineshapes are typical for multilammellar lipid bilayers in their liquid‐crystalline phase [13]. The high‐field maximum of the powder pattern corresponds to the molecules oriented perpendicular to the magnetic field axis, and the downfield maximum corresponds to the molecules parallel to the magnetic field [11]. If the molecular motional processes are restricted, an increase in the width of the NMR powder pattern is expected due to a reduction in motional averaging. In Figure 20, the first observed decrease in chemical shift anisotropy occurs around 130C, the temperature where the membrane undergoes its first pre‐transition according to the DSC profile (13.10C). In the gel state, the molecules are less flexible than in the ripple state. After the main transition at 230C, a second decrease in chemical shift values occurs, as also clearly visible in the spectral width of the wideline NMR spectrum as seen in Figure 17 (left). The smaller spectral width obtained in the liquid‐crystalline phase can be explained by an additional wobbling motion of the polar head group and the lipid molecule along its long axis [17]. The higher the temperature, the faster the rotation and the wobbling (including amplitude) of phospholipids molecules will be; all effects resulting in reduced chemical shift anisotropy and reduced width line NMR spectra.
For DMPC bilayers in the presence of 10%PazePC at pH 7.4, two different membrane domains can be observed, indicating an OxLi‐poor (broad sub spectrum) and OxLi‐rich domains, as seen in Figure 8. These two domains are also visible as a sharp and a broad component in the DSC thermogram. The P nucleus in the two domains may experience two different environment with different dynamics and perhaps slightly different magnetic environment (e.g. ox lipids with charged carboxyl group sticking out of the membrane [18] and others with neutral carboxyl group being incorporated into the hydrophobic membrane core.
Upon combining DSC and NMR data one can conclude that the two phase NMR spectra appear in a temperature range which corresponds to OxLi‐rich domains. The MAS experiment at 350C shows one isotropic peak at ‐1.0 ppm, which slightly more high‐field shifted than the one is observed for pure DMPC bilayers (Table 6).
Figure 21 shows changes in the corresponding high‐field edge chemical shift values obtained from the temperature dependent NMR spectra acquired for DMPC/10% PazePC vesicles. A correlation between the peaks with reduced values (smaller sub‐spectra) and the main peaks (outer peak) can be seen. In general, the anisotropic chemical shifts are highest at the gel‐liquid crystalline phase transition, and at high temperatures (350C). The increasing in chemical shifts can be due to two effects: orientation and dynamics of the lipid head group. About orientation, literature has shown
31
that there is reversal in the sn‐2 acyl chain of oxidized lipids [19], [20] as can be illustrated in Figure 29. The oxidized sn‐2 chain with the carboxylic functional group in PazePC can stick out of the membrane surface to be perpendicular or parallel to the bilayer [20]. Once pointing out, the negative charge may have electrostatic interactions with the positively charged nitrogen of the choline group and, hence, changes the orientation of the head group. This way the averaging of the chemical shift 31P tensor around the lipid long axis will be affected, leading to a change in observed chemical shift anisotropy. In the case of 10% PazePC present the chemical shift anisotropy of the NMR lineshapes is reduced compared to the one obtained for pure DMPC bilayers under the same conditions, as seen in Figure 13. On the other hand, the head group may be oriented closer to the magic angle with respect to the membrane surface (600 values for pure DMPC vesicles at 308 K) [13].
The reversal of oxidized sn‐2 chains can affect dynamic of the head group [20]. The oxidatively modified fatty acyl chains prefer to locate closer to the interface than the corresponding unoxidized chains to minimize the free energy penalty caused by burying the polar groups within the hydrophobic membrane core, and thereby to induce lateral expansion of the membrane [18].
The reorientation of sn‐2 chains can be illustrated in Figure 24. This expanding may lead to increasing of membrane flexibility and then faster lateral diffusion in membrane.
With DMPC vesicles containing 2 mole% of PazePC, the sample seems to be unstable, and there were additional components developed in the static NMR experiments (Figure 19). For 5% PazePC, the membrane behavior was similar (data not shown). After all the static 31P experiments, MAS NMR was run for the sample 2% and 5% PazePC. The results show that some new components occurred in the spectra over time.
At a more basic environment (pH 9.5) the membrane behavior does not generate longer a NMR powder pattern typical for pure DMPC bilayers (Figure 8, red line). The carboxylic group in PazePC molecules is totally deprotonated at this pH, and the sn‐2 acyl chain may point out of the membrane surface. That disturbs the membrane structure, and the bilayer is not formed. In addition, under this conditions the enzymatic cleavage of the fatty acids from the glycerol backbone might occur, as visible in additional components in the static (Figure 19B) and even MAS spectra.
The presence of 2 mole% PGPC shows no significant effect in solid state spectra NMR of the mixed DMPC bilayers (Figure 24). The only change is the chemical shifts of the powder patter edge. In the presence of PGPC, the chemical shifts are less negative than pure DMPC, indicating a more flexible structure in the lipid bilayer (Figure 25).
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Figure 29: A Schematic illustrations of the possible arrangements of the sn‐2 acyl chains of DMPC and PazePC. For PazePC the sn‐2 chains tend to be located close to the bilayer surface (A) or protrude out of the monolayer plane (B). Adapted from [187]
Difference between H2O and D2O as Solvents for DMPC/OxLis in NMR Experiments
Figure 23 shows the effect of the substitution of D2O by H2O on the NMR spectra of DMPC/10mole% PazePC vesicles. Clearly, the occurrence of two sub‐spectra in NMR powder pattern of DMPC/10%PazePC mixture appears now at a lower temperature range. This may imply a more ordered structure in D2O‐hydrated bilayer.
Heavy water (D2O) has different physicochemical properties to some extent compared to normal water. It has been shown that exchange of H2O by D2O affects the time‐averaged properties of the PC bilayer [21]. The substituting D2O for H2O is known to influence the interfacial region and affect the phase behavior of phosphatidylcholine. By molecular dynamic simulation, authors concluded that in D2O the bilayer surface area is slightly decreased, resulting in a denser packing of atoms in the center of the acyl chain, a higher degree of acyl chain order, and increased thickness [21]. The more ordered structure in D2O‐hydrated bilayers may cause the splitting to appear sooner in NMR experiment compared to H2O‐hydrated bilayer.
It could also been shown by DSC that in D2O, both the pretransition and main trainsition of DMPC are shifted to higher temperatures. This observation clearly supports the conclusion that the DMPC membrane possesses a more ordered structure when hydrated by D2O.
Interplay between Aβ Peptide and Oxidized lipids Containing Membranes
The Aβ peptide has shown some effects on membrane structure which contains oxidized lipids species. 31P (Figure 27) and 14N NMR (Figure 28) gives information about the lipid head group and orientation upon addition of the peptide. The smaller powder pattern in both 31P and 14N spectra at transition temperature indicating either more flexible structure of the lipid head group or there are changes in the head group orientation. It has been shown that Aβ peptide can interact with the
33
membrane interface region e.g. via its charged Lys 29 by electrostatic interactions. This type of anchoring process at the interface may affect the head group orientation.
At around transition temperature, the smaller powder pattern in 31P NMR spectra may be due to changing in head group orientation upon the presence of the Aβ peptide. If the head group position comes closer to the magic angle, it is rotating faster, results in smaller chemical shift anisotropy. The splitting in the powder patter is similar to the mixture without peptide incorporation; that indicates the binding of peptide to membrane surface does not affect the phase structure in the membrane.
These effects also appear in 14N NMR spectra. Moreover, at 230C, the powder pattern changes significantly compared to the DMPC/10%PazePC mixture behavior. That means there are significant changes in the N nucleus position upon incorporation of the Aβ peptide.
Conclusion
The results from this work lead to important conclusions. Some oxidized lipid species at certain concentrations have a strong effect on DMPC bilayers. They affect phase behavior of lipid dispersions in term of temperature and enthalpy of the transition; and the effects depend on pH of the environment and oxidized lipids concentration. At high pH the transition can be abolished indicating the bilayers structure is strongly disturbed. Crowding agents favor the more ordered state; it means the gel state is stabilized. Both DSC and NMR data show that oxidized lipids induce two phases in the DMPC dispersions at gel‐to‐liquid crystalline transition temperature, named oxidized lipid‐poor and oxidized lipid‐rich domains. The phase transition of bilayer structure also depends on solvent used; normal water and heavy water affect the behavior of DMPC/PazePC membrane differently. The final conclusion is the effect of oxidized lipids on Aβ peptide‐membrane interactions. In the presence of PazePC the membrane structure under interaction with the peptide has changed in either head group orientation and/or dynamical behavior, which can alter the physical‐chemical properties of the cell membrane.
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Acknowledgements
Firstly, I would like to thank my supervisor, Professor Gerhard Gröbner, for offering me a great opportunity to work with NMR spectroscopy, one of my most interesting subjects. During five months, he has given me a very useful guidance to improve my knowledge as well as my skill in the lab. I am appreciated to be one of his students.
I also thank for Professor Magnus Wolf‐Watz and Professor Pernilla Wittung‐Stafshede for their precious review on my work.
I also wish to thank Marcus Wallgren, for his kindness and willing help whenever I need.
For these other people, I am grateful to know them, since
Johan Vestergren did help me a lot with Origin software and is a very friendly friend,
Tofeeq Rehman instructed me about DSC software and a lot of extra help in the lab,
Tobias Sparrman offered me useful knowledge about NMR, and any help related to NMR spectroscopy,
Jan Procek has shown me many other things beside scientific knowledge, and
My family and all of my friends have been by my side, given my best advice and loved me always.
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