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1.
Sci Rep ; 10(1): 12302, 2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32704045

RESUMO

Antimicrobial resistance is a major threat to public health. Although many commercial sanitisers contain a combination of cationic surfactants and aromatic alcohols, the physical mechanisms where these two substances bind to or how they disturb bacterial membranes are still largely unknown. In this study, we designed a well-defined model of Gram-negative bacteria surfaces based on the monolayer of lipopolysaccharides with uniform saccharide head groups. Since commonly used X-ray reflectivity is sensitive to changes in the thickness, roughness and electron density but is not sensitive to elements, we employed grazing incidence X-ray fluorescence. In the absence of Ca2+, cationic surfactants can penetrate into the membrane core with no extra support by disturbing the layer of K+ coupled to negatively charged saccharide head group at z = 17 Å from the air/chain interface. On the other hand, Ca2+ confined at z = 19 Å crosslink charged saccharides and prevent the incorporation of cationic surfactants. We found that the addition of nonlethal aromatic alcohols facilitate the incorporation of cationic surfactants by the significant roughening of the chain/saccharide interface. Combination of precise localisation of ions and molecular-level structural analysis quantitatively demonstrated the synegtestic interplay of ingredients to achieve a high antibacterial activity.

2.
Chemphyschem ; 20(13): 1698-1705, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31083773

RESUMO

Semifluorinated alkanes FnHm self-assemble into nanometer-sized surface micelles at the air-water interface. In this study, we investigated how an atmosphere enriched with perfluorohexane (PFH) influences the interfacial viscoelasticity and structural order of a monolayer of FnHm by the combination of dilational rheology and grazing-incidence small-angle X-ray scattering (GISAXS). The monolayers behaved predominantly elastic which can be attributed to the strong dipole repulsions of the surface domains. Enrichment of the atmosphere with PFH lead to an increase of the compressibility and a decrease of the elastic modulus without altering the structural ordering of the FnHm molecules into highly correlated nanodomains, suggesting the adsorption of PFH molecules to the free spaces between the domains. The capability of FnHm domains to retain the structural integrity in the presence of PFH gas is promising for the fabrication of stable microbubbles for sonographic imaging.

3.
Chemphyschem ; 20(6): 898-904, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30570207

RESUMO

The structure and lateral correlation of fluorocarbon-hydrocarbon tetrablock di(F10Hm) domains at the air/water interface have been determined by quantitative analysis of grazing incidence small-angle X-ray scattering (GISAXS) data. The measured GISAXS signals can be well represented by the full calculation of the form and structure factors. The form factor suggests that di(F10Hm) domains take a hemiellipsoid shape. Both major and minor axes of the hemiellipsoids monotonically increased in response to the elongation of the hydrocarbon blocks, which can be explained by the concominant increase in van der Waals interaction. The structure factor calculated from the GISAXS signals suggests that the domains take an orthorhombic lattice. Remarkably, the lateral correlation can reach over a distance that is more than 14 times longer than the distance to the nearest neighbors. Our data suggest that quantitative GISAXS enables the optimal design of mesoscopic self-assemblies at the air/water interface by fine-tuning of the structures of molecular building blocks.

4.
J Am Chem Soc ; 140(36): 11261-11266, 2018 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-30119598

RESUMO

Water molecules confined in a nanocavity possess distinctly different characteristics from those in bulk, yet the preparation of such nanocavities is still a major experimental challenge. We report here a self-assembled vesicle of an anionic perfluoroalkylated [60]fullerene, unique for its outstanding stability and water tightness, containing water not bound to the membranes. Small-angle neutron scattering revealed that a vesicle of 14 nm outer radius contains a 2 nm thick fullerene bilayer, inside of which is a 3 nm thick membrane-bound water and unbound water in the 4 nm innermost cavity. The vesicle shows astonishingly low water permeability that is 6 to 9 orders of magnitude smaller than that of a lipid vesicle. As a result, a single vesicle isolated on a substrate can retain the interior water in air or even under high vacuum, indicating that the vesicle cavity provides a new tool for physicochemical studies of confined water as well as ions and molecules dissolved in it.

5.
Sci Rep ; 7(1): 14081, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29074975

RESUMO

Lensless, coherent X-ray diffraction microscopy has been drawing considerable attentions for tomographic imaging of whole human cells. In this study, we performed cryogenic coherent X-ray diffraction imaging of human erythrocytes with and without malaria infection. To shed light on structural features near the surface, "ghost cells" were prepared by the removal of cytoplasm. From two-dimensional images, we found that the surface of erythrocytes after 32 h of infection became much rougher compared to that of healthy, uninfected erythrocytes. The Gaussian roughness of an infected erythrocyte surface (69 nm) is about two times larger than that of an uninfected one (31 nm), reflecting the formation of protein knobs on infected erythrocyte surfaces. Three-dimensional tomography further enables to obtain images of the whole cells with no remarkable radiation damage, whose accuracy was estimated using phase retrieval transfer functions to be as good as 64 nm for uninfected and 80 nm for infected erythrocytes, respectively. Future improvements in phase retrieval algorithm, increase in degree of coherence, and higher flux in combination with complementary X-ray fluorescence are necessary to gain both structural and chemical details of mesoscopic architectures, such as cytoskeletons, membraneous structures, and protein complexes, in frozen hydrated human cells, especially under diseased states.


Assuntos
Eritrócitos/patologia , Malária/diagnóstico por imagem , Malária/patologia , Microscopia/métodos , Tomografia por Raios X/métodos , Difração de Raios X/métodos , Células Cultivadas , Criopreservação , Eritrócitos/metabolismo , Humanos , Imageamento Tridimensional/métodos , Malária/metabolismo , Proteínas/metabolismo , Tomografia por Raios X/instrumentação
6.
Chemphyschem ; 18(19): 2791-2798, 2017 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-28497902

RESUMO

The shape and size of self-assembled mesoscopic surface domains of fluorocarbon-hydrocarbon (FnHm) diblocks and the lateral correlation between these domains were quantitatively determined from grazing incidence small-angle X-ray scattering (GISAXS). The full calculation of structure and form factors unravels the influence of fluorocarbon and hydrocarbon block lengths on the diameter and height of the domains, and provides the inter-domain correlation length. The diameter of the domains, as determined from the form factor analysis, exhibits a monotonic increase in response to the systematic lengthening of each block, which can be attributed to the increase in van der Waals attraction between molecules. The pair correlation function in real space calculated from the structure factor implies that the inter-domain correlation can reach a distance that is over 25 times larger than the domain's size. The full calculation of the GISAXS signals introduced here opens a potential towards the hierarchical design of mesoscale domains of self-assembled small organic molecules, covering several orders of magnitude in space.

7.
Biochim Biophys Acta Biomembr ; 1859(5): 959-965, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28212861

RESUMO

The accumulation of phosphatidylcholine (PC) in the intestinal mucus layer is crucial for the protection of colon epithelia from the bacterial attack. It has been reported that the depletion of PC is a distinct feature of ulcerative colitis. Here we addressed the question how PC interacts with its binding proteins, the mucins, which may establish the hydrophobic barrier against colonic microbiota. In the first step, the interactions of dioleoylphosphatidylcholine (DOPC) with two mucin preparations from porcine stomach, have been studied using dynamic light scattering, zeta potential measurement, and Langmuir isotherms, suggesting that mucin binds to the surface of DOPC vesicles. The enthalpy of mucin-PC interaction could be determined by isothermal titration calorimetry. The high affinity to PC found for both mucin types seems reasonable, as they mainly consist of mucin 2, a major constituent of the flowing mucus. Moreover, by the systematic variation of net charges, we concluded that the zwitterionic DOPC has the strongest binding affinity that cannot be explained within the electrostatic interactions between charged molecules.


Assuntos
Mucosa Intestinal/metabolismo , Fosfatidilcolinas/metabolismo , Animais , Luz , Mucinas/metabolismo , Fosfatidilcolinas/química , Espalhamento de Radiação , Eletricidade Estática , Suínos
8.
Langmuir ; 33(7): 1646-1654, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28125880

RESUMO

The behavior of compound 2 [1,3,5-tri(2,2'-bipyridin-5-yl)benzene] with three bipyridine units arranged in a star geometry is investigated in the presence and absence of Ni(ClO4)2. Its properties at the air-water interface as well as after transfer onto a solid substrate are studied by several techniques including Brewster angle microscopy, X-ray reflectivity, neutron reflectivity, X-ray photoelectron spectroscopy, Rutherford backscattering spectrometry, and atomic force microscopy combined with optical microscopy. It is found that compound 2 within the monolayers formed stays almost vertical at the interface and that at high Ni2+/2 (Ni2+/2 = 4000, 20'000) ratios two of the three bipyridine units of 2 are complexed, resulting in supramolecular sheets that are likely composed of arrays of linear metal-organic complexation polymers.

9.
Nat Commun ; 7: 12870, 2016 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-27641538

RESUMO

The protein density in biological membranes can be extraordinarily high, but the impact of molecular crowding on the diffusion of membrane proteins has not been studied systematically in a natural system. The diversity of the membrane proteome of most cells may preclude systematic studies. African trypanosomes, however, feature a uniform surface coat that is dominated by a single type of variant surface glycoprotein (VSG). Here we study the density-dependence of the diffusion of different glycosylphosphatidylinositol-anchored VSG-types on living cells and in artificial membranes. Our results suggest that a specific molecular crowding threshold (MCT) limits diffusion and hence affects protein function. Obstacles in the form of heterologous proteins compromise the diffusion coefficient and the MCT. The trypanosome VSG-coat operates very close to its MCT. Importantly, our experiments show that N-linked glycans act as molecular insulators that reduce retarding intermolecular interactions allowing membrane proteins to function correctly even when densely packed.


Assuntos
Glicoproteínas Variantes de Superfície de Trypanosoma/fisiologia , Glicosilação , Glicosilfosfatidilinositóis/metabolismo , Trypanosoma
10.
J Phys Chem B ; 119(30): 9787-94, 2015 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-26125947

RESUMO

The influence of phospholipid oxidization of floating monolayers on the structure perpendicular to the global plane and on the density profiles of ions near the lipid monolayer has been investigated by a combination of grazing incidence X-ray fluorescence (GIXF) and specular X-ray reflectivity (XRR). Systematic variation of the composition of the floating monolayers unravels changes in the thickness, roughness and electron density of the lipid monolayers as a function of molar fraction of oxidized phospholipids. Simultaneous GIXF measurements enable one to qualitatively determine the element-specific density profiles of monovalent (K(+) or Cs(+)) and divalent ions (Ca(2+)) in the vicinity of the interface in the presence and absence of two types of oxidized phospholipids (PazePC and PoxnoPC) with high spatial accuracy (±5 Å). We found the condensation of Ca(2+) near carboxylated PazePC was more pronounced compared to PoxnoPC with an aldehyde group. In contrast, the condensation of monovalent ions could hardly be detected even for pure oxidized phospholipid monolayers. Moreover, pure phospholipid monolayers exhibited almost no ion specific condensation near the interface. The quantitative studies with well-defined floating monolayers revealed how the elevation of lipid oxidization level alters the structures and functions of cell membranes.


Assuntos
Fosfolipídeos/química , Eletricidade Estática , Cálcio/química , Césio/química , Elétrons , Oxirredução , Potássio/química , Espectrometria de Fluorescência , Difração de Raios X
11.
J Chem Phys ; 142(15): 154907, 2015 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-25903910

RESUMO

The mechanical properties of multilayer stacks of Gb3 glycolipid that play key roles in metabolic disorders (Fabry disease) were determined quantitatively by using specular and off-specular neutron scattering. Because of the geometry of membrane stacks deposited on planar substrates, the scattered intensity profile was analyzed in a 2D reciprocal space map as a function of in-plane and out-of-plane scattering vector components. The two principal mechanical parameters of the membranes, namely, bending rigidity and compression modulus, can be quantified by full calculation of scattering functions with the aid of an effective cut-off radius that takes the finite sample size into consideration. The bulkier "bent" Gb3 trisaccharide group makes the membrane mechanics distinctly different from cylindrical disaccharide (lactose) head groups and shorter "bent" disaccharide (gentiobiose) head groups. The mechanical characterization of membranes enriched with complex glycolipids has high importance in understanding the mechanisms of diseases such as sphingolipidoses caused by the accumulation of non-degenerated glycosphingolipids in lysosomes or inhibition of protein synthesis triggered by the specific binding of Shiga toxin to Gb3.


Assuntos
Glicolipídeos/química , Membranas Intracelulares/química , Nêutrons , Oligossacarídeos/química , Espalhamento de Radiação , Glicolipídeos/síntese química , Humanos , Lisossomos/química , Conformação Molecular
12.
Artigo em Inglês | MEDLINE | ID: mdl-23944490

RESUMO

As a defined model of outer membranes of gram negative bacteria, we investigated the interaction of monolayers of lipopolysacchrides from Salmonella enterica rough strains R90 (LPS Ra) with natural and synthetic peptides. The fine structures perpendicular to the membrane plane and the ion distribution near the interface were determined by specular x-ray reflectivity (XRR) and grazing-incidence x-ray fluorescence (GIXF) in the presence and absence of divalent cations. The unique combination of XRR and GIXF allows for the quantitative identification of different modes of interactions in a high spatial resolution, which cannot be assessed by other experimental methods. Natural fish protamine disrupts the stratified membrane structures in the absence of Ca(2+) ions, while staying away from the membrane surface in the presence of Ca(2+) ions. In contrast, synthetic antisepsis peptide Pep 19-2.5 weakly adsorbs to the membrane and stays near the uncharged sugar units even in the absence of Ca(2+). In the presence of Ca(2+), Pep 19-2.5 can reach the negatively charged inner core without destroying the barrier capability against ions.


Assuntos
Antissepsia , Membrana Celular/metabolismo , Proteínas de Peixes/metabolismo , Peptídeos/metabolismo , Protaminas/metabolismo , Salmonella enterica/citologia , Espectrometria por Raios X , Ar , Cálcio/farmacologia , Proteínas de Peixes/química , Lipopolissacarídeos/metabolismo , Peptídeos/química , Protaminas/química , Ligação Proteica/efeitos dos fármacos , Eletricidade Estática , Água/química
13.
J Phys Chem B ; 117(17): 5002-8, 2013 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-23586470

RESUMO

We have demonstrated that the complementary combination of grazing incidence X-ray fluorescence (GIXF) with specular X-ray reflectivity (XRR) can be used to quantitatively determine the density profiles of Ni(2)(+) ions complexed with chelator headgroups as well as S atoms in recombinant proteins anchored to lipid monolayers at the air/water interface. First, we prepared phospholipid monolayers incorporating chelator lipid anchors at different molar fractions at the air/water interface. The fine-structures perpendicular to the global plane of monolayers were characterized by XRR in the presence of Ni(2)(+) ions, yielding the thickness, roughness, and electron density of the stratified lipid monolayers. X-ray fluorescence intensities from Ni Kα core levels recorded at the incidence angles below and above the critical angle of total reflection allow for the determination of the position and lateral density of Ni(2)(+) ions associated with chelator headgroups with a high spatial accuracy (±5 Å). The coupling of histidine-tagged Xenopus cadherin 11 (Xcad-11) can also be identified by changes in the fines-structures using XRR. Although fluorescence intensities from S Kα level were much weaker than Ni Kα signals, we could detect the location of S atoms in recombinant Xcad-11 proteins.


Assuntos
Histidina/química , Oligopeptídeos/química , Proteínas Recombinantes de Fusão/análise , Ar , Animais , Caderinas/análise , Caderinas/genética , Caderinas/metabolismo , Histidina/genética , Histidina/metabolismo , Íons/química , Níquel/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fosfolipídeos/química , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência , Água/química , Raios X , Xenopus , Proteínas de Xenopus/análise , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
14.
J Chem Phys ; 137(20): 204907, 2012 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-23206031

RESUMO

As a physical model of the surface of cells coated with densely packed, non-crystalline proteins coupled to lipid anchors, we functionalized the surface of phospholipid membranes by coupling of neutravidin to biotinylated lipid anchors. After the characterization of fine structures perpendicular to the plane of membrane using specular X-ray reflectivity, the same membrane was characterized by grazing incidence small angle X-ray scattering (GISAXS). Within the framework of distorted wave Born approximation and two-dimensional Percus-Yevick function, we can analyze the form and structure factors of the non-crystalline, membrane-anchored proteins for the first time. As a new experimental technique to quantify the surface density of proteins on the membrane surface, we utilized grazing incidence X-ray fluorescence (GIXF). Here, the mean intermolecular distance between proteins from the sulfur peak intensities can be calculated by applying Abelé's matrix formalism. The characteristic correlation distance between non-crystalline neutravidin obtained by the GISAXS analysis agrees well with the intermolecular distance calculated by GIXF, suggesting a large potential of the combination of GISAXS and GIXF in probing the lateral density and correlation of non-crystalline proteins displayed on the membrane surface.


Assuntos
Proteínas de Membrana/química , Modelos Biológicos , Proteínas/química , Fluorescência , Ligação Proteica , Espalhamento a Baixo Ângulo , Propriedades de Superfície , Difração de Raios X
15.
J Chem Phys ; 136(7): 075102, 2012 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-22360263

RESUMO

The functionality of proteins is governed by their dynamics. We have performed a systematic investigation on four different proteins in the far-infrared spectral region under control of the two external parameters that have the strongest influence on the dynamics, namely temperature and hydration. The absorption measurements covering the frequency range from 40 cm(-1) to 690 cm(-1) (1-20 THz) close the gap between the well-studied mid-infrared and the recent THz investigations. By preparing the proteins as free-standing films, we achieve unprecedented reproducibility. Besides a featureless slope in the THz range, we can identify absorption peaks characteristic for each protein and others common to several proteins. We fit the spectra to extract the peak positions and suggest assignments for them. The far-infrared absorption spectra of all proteins are basically independent on hydration. By a detailed analysis of the sorption isotherms this can be explained by the low absorption of biological water, which resembles more the behavior of ice than that of liquid water.


Assuntos
Proteínas/química , Água/química , Absorção , Espectrofotometria Infravermelho/métodos , Temperatura
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