Lipid Phase Control and Secondary Structure of Viral Fusion Peptides Anchored in Monoolein Membranes.

The fusion of lipid membranes involves major changes of the membrane curvatures and is mediated by fusion proteins that bind to the lipid membranes. For a better understanding of the way fusion proteins steer this process, we have studied the interaction of two different viral fusion peptides, HA2-FP and TBEV-FP, with monoolein mesophases as a function of temperature and pressure at limited hydration. The fusion peptides are derived from the influenza virus hemagglutinin fusion protein (HA2-FP) and from the tick-borne encephalitis virus envelope glycoprotein E (TBEV-FP). By using synchrotron X-ray diffraction, the changes of the monoolein phase behavior upon binding the peptides have been determined and the concomitant secondary structures of the peptides have been analyzed by FTIR spectroscopy. As main results we have found that the fusion peptides interact differently with monoolein and change the pressure and temperature dependent lipid phase behavior to different extents. However, they both destabilize the fluid lamellar phase and favor phases with negative curvature, i.e. inverse bicontinuous cubic and inverse hexagonal phases. These peptide-induced phase changes can partially be reversed by the application of high pressure, demonstrating that the promotion of negative curvature is achieved by a less dense packing of the monoolein membranes by the fusion peptides. Pressure jumps across the cubic-lamellar phase transition reveal that HA2-FP has a negligible effect on the rates of the cubic and the lamellar phase formation. Interestingly, the secondary structures of the fusion peptides appear unaffected by monoolein fluid-fluid phase transitions, suggesting that the fusion peptides are the structure dominant species in the fusion process of lipid membranes.

The effects of pressure and temperature on the energetics and pivotal surface in a monoacylglycerol/water gyroid inverse bicontinuous cubic phase.

We have studied the effect of pressure and temperature on the location of the pivotal surface in a lipid inverse bicontinuous gyroid cubic phase (Q(G)(II)), described by the area at the pivotal surface (An), the volume between the pivotal surface and the bilayer midplane (Vn), and the molecular volume of the lipid (V). Small angle X-ray scattering (SAXS) was used to measure the swelling behaviour of the lipid, monolinolein, as a function of pressure and temperature, and the data were fitted to two different geometric models: the parallel interface model (PIM), and the constant mean curvature model (CMCM). The results show that an increase in temperature leads to a shift in the location of the pivotal surface towards the bilayer midplane, whilst an increase in pressure causes the pivotal surface to move towards the interfacial region. In addition, we describe the relevance of An, Vn and V for modeling the energetics of curved mesophases with specific reference to the mean curvature at the pivotal surface and discuss the significance of this parameter for modelling the energetics of curved mesophases.

Hydrostatic pressure effects on the lamellar to gyroid cubic phase transition of monolinolein at limited hydration.

Monoacylglycerol based lipids are highly important model membrane components and attractive candidates for drug encapsulation and as delivery agents. However, optimizing the properties of these lipids for applications requires a detailed understanding of the thermodynamic factors governing the self-assembled structures that they form. Here, we report on the effects of hydrostatic pressure, temperature, and water composition on the structural behavior and stability of inverse lyotropic liquid crystalline phases adopted by monolinolein (an unsaturated monoacylglycerol having cis-double bonds at carbon positions 9 and 12) under limited hydration conditions. Six pressure-temperature phase diagrams have been determined using small-angle X-ray diffraction at water contents between 15 wt % and 27 wt % water, in the range 10-40 °C and 1-3000 bar. The gyroid bicontinuous cubic (Q(II)(G)) phase is formed at low pressure and high temperatures, transforming to a fluid lamellar (L(α)) phase at high pressures and low temperature via a region of Q(II)(G)/L(α) coexistence. Pressure stabilizes the lamellar phase over the Q(II)(G) phase; at fixed pressure, increasing the water content causes the coexistence region to move to lower temperature. These trends are consistent throughout the hydration range studied. Moreover, at fixed temperature, increasing the water composition increases the pressure at which the Q(II)(G) to L(α) transition takes place. We discuss the qualitative effect of pressure, temperature, and water content on the stability of the Q(II)(G) phase.

Structure and phase behavior of archaeal lipid monolayers.

We report X-ray reflectivity (XRR) and grazing incidence X-ray diffraction (GIXD) measurements of archaeal bipolar tetraether lipid monolayers at the air-water interface. Specifically, Langmuir films made of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at three different temperatures, i.e., 68, 76, and 81 °C, were examined. The dependence of the structure and packing properties of PLFE monolayers on surface pressure were analyzed in a temperature range between 10 and 50 °C at different pH values. Additionally, the interaction of PLFE monolayers (using lipids derived from cells grown at 76 °C) with the ion channel peptide gramicidin was investigated as a function of surface pressure. A total monolayer thickness of approximately 30 Å was found for all monolayers, hinting at a U-shaped conformation of the molecules with both head groups in contact with the interface. The monolayer thickness increased with rising film pressure and decreased with increasing temperature. At 10 and 20 °C, large, highly crystalline domains were observed by GIXD, whereas at higher temperatures no distinct crystallinity could be observed. For lipids derived from cells grown at higher temperatures, a slightly more rigid structure in the lipid dibiphytanyl chains was observed. A change in the pH of the subphase had an influence only on the structure of the lipid head groups. The addition of gramicidin to an PLFE monolayer led to a more disordered state as observed by XRR. In GIXD measurements, no major changes in lateral organization could be observed, except for a decrease of the size of crystalline domains, indicating that gramicidin resides mainly in the disordered areas of the monolayer and causes local membrane perturbation, only.

Effects of specific versus nonspecific ionic interactions on the structure and lateral organization of lipopolysaccharides.

We report x-ray reflectivity and grazing incidence x-ray diffraction measurements of lipopolysaccharide (LPS) monolayers at the water-air interface. Our investigations reveal that the structure and lateral ordering of the LPS molecules is very different from phospholipid systems and can be modulated by the ionic strength of the aqueous subphase in an ion-dependent manner. Our findings also indicate differential effects of monovalent and divalent ions on the two-dimensional ordering of lipid domains. Na(+) ions interact unspecifically with LPS molecules based on their ability to efficiently screen the negative charges of the LPS molecules, whereas Ca(2+) ions interact specifically by cross-linking adjacent molecules in the monolayer. At low lateral pressures, Na(+) ions present in the subphase lead to a LPS monolayer structure ordered over large areas with high compressibility, nearly hexagonal packing of the hydrocarbon chains, and high density in the LPS headgroup region. At higher film pressures, the LPS monolayer becomes more rigid and results in a less perfect, oblique packing of the LPS hydrocarbon chains as well as a smaller lateral size of highly ordered domains on the monolayer. Furthermore, associated with the increased surface pressure, a conformational change of the sugar headgroups occurs, leading to a thickening of the entire LPS monolayer structure. The effect of Ca(2+) ions in the subphase is to increase the rigidity of the LPS monolayer, leading to an oblique packing of the hydrocarbon chains already at low film pressures, an upright orientation of the sugar moieties, and much smaller sizes of ordered domains in the plane of the monolayer. In the presence of both Na(+)- and Ca(2+) ions in the subphase, the screening effect of Na(+) is predominant at low film pressures, whereas, at higher film pressures, the structure and lateral organization of LPS molecules is governed by the influence of Ca(2+) ions. The unspecific charge-screening effect of the Na(+) ions on the conformation of the sugar moiety becomes less dominant at biologically relevant lateral pressures.

High pressure cell for neutron reflectivity measurements up to 2500 bar.

The design of a high pressure (HP) cell for neutron reflectivity experiments is described. The cell can be used to study solid-liquid interfaces under pressures up to 2500 bar (250 MPa). The sample interface is based on a thick silicon block with an area of about 14 cm(2). This area is in contact with the sample solution which has a volume of only 6 cm(3). The sample solution is separated from the pressure transmitting medium, water, by a thin flexible polymer membrane. In addition, the HP cell can be temperature-controlled by a water bath in the range 5-75°C. By using an aluminum alloy as window material, the assembled HP cell provides a neutron transmission as high as 41%. The maximum angle of incidence that can be used in reflectivity experiments is 7.5°. The large accessible pressure range and the low required volume of the sample solution make this HP cell highly suitable for studying pressure-induced structural changes of interfacial proteins, supported lipid membranes, and, in general, biomolecular systems that are available in small quantities, only. To illustrate the performance of the HP cell, we present neutron reflectivity data of a protein adsorbate under high pressure and a lipid film which undergoes several phase transitions upon pressurization.

Structural plasticity of staphylococcal nuclease probed by perturbation with pressure and pH.

The ionization of internal groups in proteins can trigger conformational change. Despite this being the structural basis of most biological energy transduction, these processes are poorly understood. Small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy experiments at ambient and high hydrostatic pressure were used to examine how the presence and ionization of Lys-66, buried in the hydrophobic core of a stabilized variant of staphylococcal nuclease, affect conformation and dynamics. NMR spectroscopy at atmospheric pressure showed previously that the neutral Lys-66 affects slow conformational fluctuations globally, whereas the effects of the charged form are localized to the region immediately surrounding position 66. Ab initio models from SAXS data suggest that when Lys-66 is charged the protein expands, which is consistent with results from NMR spectroscopy. The application of moderate pressure (<2 kbar) at pH values where Lys-66 is normally neutral at ambient pressure left most of the structure unperturbed but produced significant nonlinear changes in chemical shifts in the helix where Lys-66 is located. Above 2 kbar pressure at these pH values the protein with Lys-66 unfolded cooperatively adopting a relatively compact, albeit random structure according to Kratky analysis of the SAXS data. In contrast, at low pH and high pressure the unfolded state of the variant with Lys-66 is more expanded than that of the reference protein. The combined global and local view of the structural reorganization triggered by ionization of the internal Lys-66 reveals more detectable changes than were previously suggested by NMR spectroscopy at ambient pressure.

High-pressure SAXS study of folded and unfolded ensembles of proteins.

A structural interpretation of the thermodynamic stability of proteins requires an understanding of the structural properties of the unfolded state. High-pressure small-angle x-ray scattering was used to measure the effects of temperature, pressure, denaturants, and stabilizing osmolytes on the radii of gyration of folded and unfolded state ensembles of staphylococcal nuclease. A set of variants with the internal Val-66 replaced with Ala, Tyr, or Arg was used to examine how changes in the volume and polarity of an internal microcavity affect the dimensions of the native state and the pressure sensitivity of the ensemble. The unfolded state ensembles achieved for these proteins with high pressure were more compact than those achieved at high temperature, and were all very sensitive to the presence of urea and glycerol. Substitutions at the hydrophobic core detectably altered the conformation of the protein, even in the folded state. The introduction of a charged residue, such as Arg, inside the hydrophobic interior of a protein could dramatically alter the structural properties, even those of the unfolded state. The data suggest that a charge at an internal position can interfere with the formation of transient hydrophobic clusters in the unfolded state, and ensure that the pressure-unfolded form of a protein occupies the maximum volume possible. Only at high temperatures does the radius of gyration of the unfolded state ensemble approach the value for a statistical random coil.

Unique features of the folding landscape of a repeat protein revealed by pressure perturbation.

The volumetric properties of proteins yield information about the changes in packing and hydration between various states along the folding reaction coordinate and are also intimately linked to the energetics and dynamics of these conformations. These volumetric characteristics can be accessed via pressure perturbation methods. In this work, we report high-pressure unfolding studies of the ankyrin domain of the Notch receptor (Nank1-7) using fluorescence, small-angle x-ray scattering, and Fourier transform infrared spectroscopy. Both equilibrium and pressure-jump kinetic fluorescence experiments were consistent with a simple two-state folding/unfolding transition under pressure, with a rather small volume change for unfolding compared to proteins of similar molecular weight. High-pressure fluorescence, Fourier transform infrared spectroscopy, and small-angle x-ray scattering measurements revealed that increasing urea over a very small range leads to a more expanded pressure unfolded state with a significant decrease in helical content. These observations underscore the conformational diversity of the unfolded-state basin. The temperature dependence of pressure-jump fluorescence relaxation measurements demonstrated that at low temperatures, the folding transition state ensemble (TSE) lies close in volume to the folded state, consistent with significant dehydration at the barrier. In contrast, the thermal expansivity of the TSE was found to be equivalent to that of the unfolded state, indicating that the interactions that constrain the folded-state thermal expansivity have not been established at the folding barrier. This behavior reveals a high degree of plasticity of the TSE of Nank1-7.

Microdomains in lipid vesicles: structure and distribution assessed by small-angle neutron scattering.

We examined the structure and distribution of microdomains in ternary multilamellar and unilamellar vesicles representing a canonical model raft mixture consisting of dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and cholesterol via contrast matched small-angle neutron scattering (SANS). The impetus of the work was to reveal the size and distribution of microdomains consistent with the form factor and intermolecular structure factor S(Q) of the scattering pattern of unilamellar and multilamellar vesicles prepared from this ternary lipid system. The data are consistent with the presence of cylindrically shaped microdomains with an average radius of approximately 15 nm assembled in a fractal-like geometry, and with corresponding modeling studies. In the multilamellar vesicle system, coupling of domains across the interlamellar water layer is observed.

Elucidating the mechanism of lipid membrane-induced IAPP fibrillogenesis and its inhibition by the red wine compound resveratrol: a synchrotron X-ray reflectivity study.

The islet amyloid polypeptide (IAPP) or amylin is a pancreatic hormone and crucially involved in the pathogenesis of type-II diabetes mellitus (T2DM). Aggregation and amyloid formation of IAPP is considered as the primary culprit for pancreatic beta-cell loss in T2DM patients. In this study, first X-ray reflectivity (XRR) measurements on IAPP at lipid interfaces have been carried out, providing a molecular level characterization of the first steps of the lipid-induced fibrillation process of IAPP, which is initiated by lipid-induced nucleation, oligomerization, followed by detachment of larger IAPP aggregate structures from the lipid membrane, and terminated by the formation of mature fibrils in the bulk solution. The adsorption process of IAPP at lipid interfaces in the absence and presence of negatively charged lipid has also been studied by complementary ATR-FTIR spectroscopic measurements. The morphological properties were followed by atomic force microscopy (AFM). Moreover, we show that the polyphenolic red wine compound resveratrol is able to inhibit IAPP aggregation also in the presence of aggregation-fostering negatively charged lipid interfaces, revealing its potential as a drug candidate for T2DM.

Marasmius scorodonius extracellular dimeric peroxidase - exploring its temperature and pressure stability.

The temperature and pressure dependent stability and function of MsP1, an uncommon peroxidase from the basidiomycetous fungus Marasmius scorodonius were investigated. To this end, a series of biophysical techniques (DSC, fluorescence and FTIR spectroscopy, small-angle X-ray scattering) were combined with enzymatic studies of the enzyme. The dimeric MsP1 turned out to be not only rather thermostable, but also highly resistant to pressure, i.e., up to temperatures of about 65 degrees C and pressures as high as 8-10 kbar at ambient temperatures. Remarkably, the activity of MsP1 increased by a factor of two until approximately 500 bar. At about 2 kbar, the enzymatic activity was still as high as under ambient pressure conditions. As revealed by the fluorescence and SAXS data, the increased activity of MsP1 at pressures around 500 bar may result from slight structural changes, which might stabilize the transition state of the enzymatic reaction. Owing to this marked high pressure stability of MsP1, it may represent a valuable tool for industrial high pressure applications.

Interaction of IAPP and insulin with model interfaces studied using neutron reflectometry.

The islet amyloid polypeptide (IAPP) and insulin are coproduced by the beta-cells of the pancreatic islets of Langerhans. Both peptides can interact with negatively charged lipid membranes. The positively charged islet amyloid polypeptide partially inserts into these membranes and subsequently forms amyloid fibrils. The amyloid fibril formation of insulin is also accelerated by the presence of negatively charged lipids, although insulin has a negative net charge at neutral pH-values. We used water-polymer model interfaces to differentiate between the hydrophobic and electrostatic interactions that can drive these peptides to adsorb at an interface. By applying neutron reflectometry, the scattering-length density profiles of IAPP and insulin, as adsorbed at three different water-polymer interfaces, were determined. The islet amyloid polypeptide most strongly adsorbed at a hydrophobic poly-(styrene) surface, whereas at a hydrophilic, negatively charged poly-(styrene sulfonate) interface, the degree of adsorption was reduced by 50%. Almost no IAPP adsorption was evident at this negatively charged interface when we added 100 mM NaCl. On the other hand, negatively charged insulin was most strongly attracted to a hydrophilic, negatively charged interface. Our results suggest that IAPP is strongly attracted to a hydrophobic surface, whereas the few positive charges of IAPP cannot warrant a permanent immobilization of IAPP at a hydrophilic, negatively charged surface at an ionic strength of 100 mM. Furthermore, the interfacial accumulation of insulin at a hydrophilic, negatively charged surface may represent a favorable precondition for nucleus formation and fibril formation.

X-ray kinematography of phase transformations of three-component lipid mixtures: a time-resolved synchrotron X-ray scattering study using the pressure-jump relaxation technique.

By using the pressure-jump relaxation technique in combination with time-resolved synchrotron small-angle X-ray diffraction (TRSAXS), the kinetics of lipid phase transformations of ternary lipid mixtures serving as model systems of heterogeneous raftlike membranes were investigated. To this end, we first established the temperature-pressure phase diagram of a model lipid raft mixture, 1,2-dioleoyl- sn-glycero-3-phosphatidylcholine (DOPC)/1,2-dipalmitoyl- sn-glycero-3-phosphatidylcholine (DPPC)/cholesterol (1:2:1), using Fourier transform infrared spectroscopy and SAXS, covering the pressure range from 1 bar to 10 kbar at temperatures in the range from 7 to 80 degrees C. We then studied the kinetics of interlamellar phase transitions of the ternary lipid system involving transitions from the fluidlike (liquid-disordered, l d) phase to the liquid-ordered (l o)/liquid-disordered (l d) two-phase coexistence region as well as between the two- and three-phase coexistence regions of the system, where also solid-ordered phases (s o) are involved. The phase transition from the all-fluid l d phase to the l o+l d two-phase coexistence region turns out to be rather rapid. Phases appear or disappear within the 25 ms time resolution of the technique, followed by a slow lattice relaxation process, which, depending on the pressure-jump amplitude, takes several seconds. Contrary to many one-component phospholipid phase transitions, the kinetics of the l d <--> l o+l d transition follows a similar time scale and mechanism for the pressurization and depressurization direction. A similar behavior is observed for the phase transition kinetics of the s o+l o+l d <--> l o+l d transformation and even for the s o+l o+l d <--> l d transformation, jumping across the l o+l d two-phase region. All transitions are fully reversible, and no intermediate states are populated. As indicated by the complex relaxation profiles observed, the overall rates observed seem to reflect the effect of coupling of various dynamical processes through the transformation, involving fast conformational changes in the sub-millisecond time regime and slow relaxation of the lattices growing, probably being largely controlled by the transport and redistribution of water into and in the new phases of the multilamellar vesicle assemblies.

The temperature-pressure phase diagram of a DPPC-ergosterol fungal model membrane -- a SAXS and FT-IR spectroscopy study.

We investigated the phase behaviour of the binary lipid mixture dipalmitoylphosphatidylcholine (DPPC)-ergosterol (78:22mol%) as a function of temperature and pressure using Fourier transform-infrared (FT-IR) spectroscopy and small-angle X-ray scattering (SAXS). A temperature range from 10 to 70 degrees C and a pressure range from 1 to approximately 8500bar was covered to be able to establish a p,T-phase diagram of this model mixture of a fungal plasma membrane. Our results show a liquid-ordered/solid-ordered (l(o)+s(o)) two-phase coexistence region up to 41 degrees C, followed by a liquid-disordered/liquid-ordered (l(d)+l(o)) coexistence region up to approximately 57.5 degrees C, giving way to an all-fluid-like l(d) phase at higher temperatures. With increasing pressure, all phase boundaries shift upwards to higher temperatures. The width of the raft-like l(d)+l(o) two-phase coexistence region is found to be about 2kbar over the temperature range covered. At higher pressures, transitions to a l(o)+s(o) two-phase region and, at still higher pressures and low temperatures, an all-ordered s(o) phase is detected. Finally, the biological (high pressure-induced upregulation of ergosterol synthesis) and biotechnological (inactivation of fungal microorganisms) relevance of these studies is discussed. The results may be particularly interesting for understanding the function of membrane proteins under external stress conditions, such as high pressure. Interestingly, in this pressure range of about 2kbar, ceasing of membrane protein function has been observed in natural membrane environments. They may be inactivated under pressure/temperature conditions when the plasma membrane becomes too rigid, i.e., reaches an overall ordered conformational state.