| 1. |
- Arleth, Lise, et al.
(author)
-
Block-copolymer micro-emulsion with solvent-induced segregation
- 2007
-
In: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 23:4, s. 2117-2125
-
Journal article (peer-reviewed)abstract
- Reverse micelle formation of the poly(ethylene oxide)/poly(propylene oxide) block-copolymer (EO)(13)(PO)(30)(EO)(13) (L64) in p-xylene was investigated as a function of water content and copolymer content, using small-angle neutron scattering (SANS). PEO/PPO block-copolymers are generally soluble in xylene but without forming aggregates. However, the effective block segregation increases dramatically upon addition of small amounts of water, and micelles form. The SANS data were analyzed using an absolute scale model fitting approach. This way, a detailed quantitative description of the system in terms of unimer concentration, micelle structure, and aggregation number as well as particle-particle interactions can be obtained. This approach throws light on very atypical features of the system as compared to standard amphiphilic systems. Data from samples measured along water-swelling lines with fixed EO/p-xylene-d(10) molar ratios show that reverse micelles are formed at the water/EO molar ratio, W-0 congruent to 0.2, independent of copolymer concentration. The majority of the block-copolymers are on a free monomer state (unimer state) at this W-0. Increasing W-0 above 0.2 only has a small effect on the micelle size. However, it does induce a strong increase of the total number of micelles and induce a corresponding decrease of the unimer concentration. On the other hand, increasing the overall copolymer concentration at fixed W-0 gives rise to a significant decrease of the micelle size in terms of the micellar aggregation number. This observed behavior is totally different from what is normally observed for binary surfactant-solvent systems and droplet micro-emulsion systems, respectively. We believe that the atypical behavior is a result of the unusually weak segregation in the system, and we are not aware of previous discussions of the phenomenon for reverse micellar systems.
|
|
| 2. |
- Fatouros, Dimitrios G., et al.
(author)
-
Structural development of self nano emulsifying drug delivery systems (SNEDDS) during In vitro lipid digestion monitored by small-angle x-ray scattering
- 2007
-
In: Pharmaceutical Research. - : Springer Science and Business Media LLC. - 1573-904X .- 0724-8741. ; 24:10, s. 1844-1853
-
Journal article (peer-reviewed)abstract
- Purpose. To investigate the structural development of the colloid phases generated during lipolysis of a lipid-based formulation in an in vitro lipolysis model, which simulates digestion in the small intestine. Materials and Methods. Small-Angle X-Ray scattering (SAXS) coupled with the in vitro lipolysis model which accurately reproduces the solubilizing environment in the gastrointestinal tract and simulates gastrointestinal lipid digestion through the use of bile and pancreatic extracts. The combined method was used to follow the intermediate digestion products of a self nano emulsified drug delivery system (SNEDDS) under fasted conditions. SNEDDS is developed to facilitate the uptake of poorly soluble drugs. Results. The data revealed that a lamellar phase forms immediately after initiation of lipolysis, whereas a hexagonal phase is formed after 60 min. The change of the relative amounts of these phases clearly demonstrates that lipolysis is a dynamic process. The formation of these phases is driven by the lipase which continuously hydrolyzes triglycerides from the oil-cores of the nanoemulsion droplets into mono- and diglycerides and fatty acids. We propose that this change of the over-all composition of the intestinal fluid with increased fraction of hydrolyzed nanoemulsion induces a change in the composition and effective critical packing parameter of the amphiphilic molecules, which determines the phase behavior of the system. Control experiments (only the digestion medium) or the surfactant (Cremophor RH 40) revealed the formation of a lamellar phase demonstrating that the hexagonal phase is due to the hydrolysis of the SNEDDS formulation. Conclusion. The current results demonstrate that SAXS measurements combined with the in vitro dynamic lipolysis model may be used to elucidate the processes encountered during the digestion of lipid-based formulations of poorly soluble drugs for oral drug delivery. Thus the combined methods may act as an efficient screening tool.
|
|
| 3. |
- Galvagnion, Celine, et al.
(author)
-
Structural characterisation of α-synuclein-membrane interactions and the resulting aggregation using small angle scattering
- 2024
-
In: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry. - 1463-9076 .- 1463-9084. ; 26:14, s. 10998-11013
-
Journal article (peer-reviewed)abstract
- The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloidogenic proteins, such as alpha-synuclein and amyloid beta, interact with lipids, and this interaction can strongly favour the formation of amyloid fibrils. In particular the primary nucleation step, i.e. the de novo formation of amyloid fibrils, has been shown to be accelerated by lipids. However, the exact mechanism of this acceleration is still mostly unclear. Here we use a range of scattering methods, such as dynamic light scattering (DLS) and small angle X-ray and neutron scattering (SAXS and SANS) to obtain structural information on the binding of alpha-synuclein to model membranes formed from negatively charged lipids and their co-assembly into amyloid fibrils. We find that the model membranes take an active role in the reaction. The binding of alpha synuclein to the model membranes immediately induces a major structural change in the lipid assembly, which leads to a break-up into small and mostly disc- or rod-like lipid-protein particles. This transition can be reversed by temperature changes or proteolytic protein removal. Incubation of the small lipid-alpha-synuclein particles for several hours, however, leads to amyloid fibril formation, whereby the lipids are incorporated into the amyloid fibrils. alpha S binding to DLPS and DMPS leads to a ms fast reversible deformation into disks and rods. Upon further incubation, lipid rods elongate within the same time scale as that of amyloid formation confirming lipids co-assembly with alpha S into fibrils.
|
|
| 4. |
- Galvagnion, Céline, et al.
(author)
-
Structural characterisation of α-synuclein-membrane interactions and the resulting aggregation using small angle scattering
- 2024
-
In: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry. - 1463-9076 .- 1463-9084. ; 26:14, s. 10998-11013
-
Journal article (peer-reviewed)abstract
- The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloidogenic proteins, such as α-synuclein and amyloid β, interact with lipids, and this interaction can strongly favour the formation of amyloid fibrils. In particular the primary nucleation step, i.e. the de novo formation of amyloid fibrils, has been shown to be accelerated by lipids. However, the exact mechanism of this acceleration is still mostly unclear. Here we use a range of scattering methods, such as dynamic light scattering (DLS) and small angle X-ray and neutron scattering (SAXS and SANS) to obtain structural information on the binding of α-synuclein to model membranes formed from negatively charged lipids and their co-assembly into amyloid fibrils. We find that the model membranes take an active role in the reaction. The binding of α synuclein to the model membranes immediately induces a major structural change in the lipid assembly, which leads to a break-up into small and mostly disc- or rod-like lipid-protein particles. This transition can be reversed by temperature changes or proteolytic protein removal. Incubation of the small lipid-α-synuclein particles for several hours, however, leads to amyloid fibril formation, whereby the lipids are incorporated into the amyloid fibrils.
|
|
| 5. |
- Hendus-Altenburger, Ruth, et al.
(author)
-
The intracellular lipid-binding domain of human Na+/H+ exchanger 1 forms a lipid-protein co-structure essential for activity
- 2020
-
In: Communications Biology. - : Nature Publishing Group. - 2399-3642. ; 3:1
-
Journal article (peer-reviewed)abstract
- Dynamic interactions of proteins with lipid membranes are essential regulatory events in biology, but remain rudimentarily understood and particularly overlooked in membrane proteins. The ubiquitously expressed membrane protein Na+/H+-exchanger 1 (NHE1) regulates intracellular pH (pHi) with dysregulation linked to e.g. cancer and cardiovascular diseases. NHE1 has a long, regulatory cytosolic domain carrying a membrane-proximal region described as a lipid-interacting domain (LID), yet, the LID structure and underlying molecular mechanisms are unknown. Here we decompose these, combining structural and biophysical methods, molecular dynamics simulations, cellular biotinylation- and immunofluorescence analysis and exchanger activity assays. We find that the NHE1-LID is intrinsically disordered and, in presence of membrane mimetics, forms a helical αα-hairpin co-structure with the membrane, anchoring the regulatory domain vis-a-vis the transport domain. This co-structure is fundamental for NHE1 activity, as its disintegration reduced steady-state pHi and the rate of pHi recovery after acid loading. We propose that regulatory lipid-protein co-structures may play equally important roles in other membrane proteins.
|
|
| 6. |
- Henriques, João, et al.
(author)
-
On the Calculation of SAXS Profiles of Folded and Intrinsically Disordered Proteins from Computer Simulations
- 2018
-
In: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836. ; 430:16, s. 2521-2539
-
Journal article (peer-reviewed)abstract
- Solution techniques such as small-angle X-ray scattering (SAXS) play a central role in structural studies of intrinsically disordered proteins (IDPs); yet, due to low resolution, it is generally necessary to combine SAXS with additional experimental sources of data and to use molecular simulations. Computational methods for the calculation of theoretical SAXS intensity profiles can be separated into two groups, depending on whether the solvent is modeled implicitly as continuous electron density or considered explicitly. The former offers reduced computational cost but requires the definition of a number of free parameters to account for, for example, the excess density of the solvation layer. Overfitting can thus be an issue, particularly when the structural ensemble is unknown. Here, we investigate and show how small variations of the contrast of the hydration shell, δρ, severely affect the outcome, analysis and interpretation of computed SAXS profiles for folded and disordered proteins. For both the folded and disordered proteins studied here, using a default δρ may, in some cases, result in the calculation of non-representative SAXS profiles, leading to an overestimation of their size and a misinterpretation of their structural nature. The solvation layer of the different IDP simulations also impacts their size estimates differently, depending on the protein force field used. The same is not true for the folded protein simulations, suggesting differences in the solvation of the two classes of proteins, and indicating that different force fields optimized for IDPs may cause expansion of the polypeptide chain through different physical mechanisms.
|
|
| 7. |
- Jønsson, Rie, et al.
(author)
-
Aggregative adherence fimbriae form compact structures as seen by SAXS
- 2023
-
In: Scientific Reports. - 2045-2322. ; 13:1
-
Journal article (peer-reviewed)abstract
- Bacterial colonization is mediated by fimbriae, which are thin hair-like appendages dispersed from the bacterial surface. The aggregative adherence fimbriae from enteroaggregative E. coli are secreted through the outer membrane and consist of polymerized minor and major pilin subunits. Currently, the understanding of the structural morphology and the role of the minor pilin subunit in the polymerized fimbriae are limited. In this study we use small-angle X-ray scattering to reveal the structural morphology of purified fimbriae in solution. We show that the aggregative fimbriae are compact arrangements of subunit proteins Agg5A + Agg3B which are assembled pairwise on a flexible string rather than extended in relatively straight filaments. Absence of the minor subunit leads to less compact fimbriae, but did not affect the length. The study provides novel insights into the structural morphology and assembly of the aggregative adherence fimbriae. Our study suggests that the minor subunit is not located at the tip of the fimbriae as previously speculated but has a higher importance for the assembled fimbriae by affecting the global structure.
|
|
| 8. |
- Luchini, Alessandra, et al.
(author)
-
Dark peptide discs for the investigation of membrane proteins in supported lipid bilayers : the case of synaptobrevin 2 (VAMP2)
- 2022
-
In: Nanoscale Advances. - : Royal Society of Chemistry. - 2516-0230. ; 10:17
-
Journal article (peer-reviewed)abstract
- Supported lipid bilayers (SLBs) are commonly used as model systems mimicking biological membranes. Recently, we reported a new method to produce SLBs with incorporated membrane proteins, which is based on the application of peptide discs [Luchini et al., Analytical Chemistry, 2020, 92, 1081-1088]. Peptide discs are small discoidal particles composed of a lipid core and an outer belt of self-assembled 18A peptides. SLBs including membrane proteins can be formed by depositing the peptide discs on a solid support and subsequently removing the peptide by buffer rinsing. Here, we introduce a new variant of the 18A peptide, named dark peptide (d18A). d18A exhibits UV absorption at 214 nm, whereas the absorption at 280 nm is negligible. This improves sample preparation as it enables a direct quantification of the membrane protein concentration in the peptide discs by measuring UV absorption at 280 nm. We describe the application of the peptide discs prepared with d18A (dark peptide discs) to produce SLBs with a membrane protein, synaptobrevin 2 (VAMP2). The collected data showed the successful formation of SLBs with high surface coverage and incorporation of VAMP2 in a single orientation with the extramembrane domain exposed towards the bulk solvent. Compared to 18A, we found that d18A was more efficiently removed from the SLB. Our data confirmed the structural organisation of VAMP2 as including both alpha-helical and beta-sheet secondary structure. We further verified the orientation of VAMP2 in the SLBs by characterising the binding of VAMP2 with alpha-synuclein. These results point at the produced SLBs as relevant membrane models for biophysical studies as well as nanostructured biomaterials.
|
|
| 9. |
- Luchini, Alessandra, et al.
(author)
-
Peptide Disc Mediated Control of Membrane Protein Orientation in Supported Lipid Bilayers for Surface-Sensitive Investigations
- 2020
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 92:1, s. 1081-1088
-
Journal article (peer-reviewed)abstract
- In vitro characterization of membrane proteins requires experimental approaches providing mimics of the microenvironment that proteins encounter in native membranes. In this context, supported lipid bilayers provide a suitable platform to investigate membrane proteins by a broad range of surface-sensitive techniques such as neutron reflectometry (NR), quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), atomic force microscopy (AFM), and fluorescence microscopy. Nevertheless, the successful incorporation of membrane proteins in lipid bilayers with sufficiently high concentration and controlled orientation relative to the bilayer remains challenging. We propose the unconventional use of peptide discs made by phospholipids and amphipathic 18A peptides to mediate the formation of supported phospholipid bilayers with two different types of membrane proteins, CorA and tissue factor (TF). The membrane proteins are reconstituted in peptide discs, deposited on a solid surface, and the peptide molecules are then removed with extensive buffer washes. This leaves a lipid bilayer with a relatively high density of membrane proteins on the support surface. As a very important feature, the strategy allows membrane proteins with one large extramembrane domain to be oriented in the bilayer, thus mimicking the in vivo situation. The method is highly versatile, and we show its general applicability by characterizing with the above-mentioned surface-sensitive techniques two different membrane proteins, which were efficiently loaded in the supported bilayers with similar to 0.6% mol/mol (protein/lipid) concentration corresponding to 35% v/v for CorA and 8% v/v for TF. Altogether, the peptide disc mediated formation of supported lipid bilayers with membrane proteins represents an attractive strategy for producing samples for structural and functional investigations of membrane proteins and for preparation of suitable platforms for drug testing or biosensor development.
|
|
| 10. |
- Luchini, Alessandra, et al.
(author)
-
Peptide discs as precursors of biologically relevant supported lipid bilayers
- 2021
-
In: Journal of Colloid and Interface Science. - : Elsevier. - 0021-9797 .- 1095-7103. ; 585, s. 376-385
-
Journal article (peer-reviewed)abstract
- Supported lipid bilayers (SLBs) are commonly used to investigate the structure and dynamics of biological membranes. Vesicle fusion is a widely exploited method to produce SLBs. However, this process becomes less favoured when the vesicles contain complex lipid mixtures, e.g. natural lipid extracts. In these cases, it is often necessary to change experimental parameters, such as temperature, to unphysiological values to trigger the SLB formation. This may induce lipid degradation and is also not compatible with including membrane proteins or other biomolecules into the bilayers. Here, we show that the peptide discs, ~10 nm discoidal lipid bilayers stabilized in solution by a self-assembled 18A peptide belt, can be used as precursors for SLBs. The characterizations by means of neutron reflectometry and attenuated total reflectance-FTIR spectroscopy show that SLBs were successfully formed both from synthetic lipid mixtures (surface coverage 90-95%) and from natural lipid mixtures (surface coverage ~85%). Traces of 18A peptide (below 0.02 M ratio) left at the support surface after the bilayer formation do not affect the SLB structure. Altogether, we demonstrate that peptide disc formation of SLBs is much faster than the SLB formation by vesicle fusion and without the need of altering any experimental variable from physiologically relevant values.
|
|
