12 research outputs found
Fine-Structures, Lateral Correlation and Diffusion of Membrane-Associated Proteins on Biological Membrane Surfaces
The primary aim of this thesis is to investigate the lateral diffusion, correlation and interactions of proteins and peptides with cell membrane models by the combination of experimental techniques in real and reciprocal space. In Chapter 4, the characteristic distance and range of lateral correlation between non-crystalline proteins anchored to fluid lipid monolayers at high surface densities were determined by grazing incidence small angle X-ray scattering (GISAXS) for the first time. Moreover, the lateral density of membrane-anchored proteins could be quantified from Sulfur Kα emission detected by grazing incidence X-ray fluorescence (GIXF). In Chapter 5, the influence of molecular crowding on the lateral diffusion of membrane-anchored proteins was investigated by fluorescence recovery after photobleaching and single particle tracking microscopy, yielding a clear transition from free diffusion to confined diffusion. In Chapter 6, the interactions between antimicrobial peptides and bacterial membrane models are probed by GIXF that allows for the identification of the spatial localization of ions with Ă
ngstrom accuracy. This enabled one to discriminate different "modes" of membrane-protein interactions, such as adsorption and incorporation on the molecular level. The obtained results demonstrated that the use of real and reciprocal space techniques can provide information about fine-structures, electrostatics, and dynamic correlation at biological interfaces
Id1 and Id3 Are Regulated Through MatrixâAssisted Autocrine BMP Signaling and Represent Therapeutic Targets in Melanoma
Impact of Lipid Oxidization on Vertical Structures and Electrostatics of Phospholipid Monolayers Revealed by Combination of Specular Xâray Reflectivity and Grazing-Incidence Xâray Fluorescence
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<sup>+</sup> or Cs<sup>+</sup>) and divalent
ions (Ca<sup>2+</sup>) 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 Ă
). We found the condensation
of Ca<sup>2+</sup> 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
Ion-Mediated Cross-linking of Biopolymers Confined at Liquid/Liquid Interfaces Probed by In Situ High-Energy Grazing Incidence Xray Photon Correlation Spectroscopy
N-glycosylation enables high lateral mobility of GPI-anchored proteins at a molecular crowding threshold
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
Dendronized oligoethylene glycols with phosphonate tweezers for cell-repellent coating of oxide surfaces: coarse-scale and nanoscopic interfacial forces
Dendronized oligoethylene glycols (dendron OEGs) with two phosphonate groups (phosphonate tweezers) have been drawing significant attention as a new class of coating materials for superparamagnetic iron oxide surfaces. However, despite dendron OEGs showing outstanding stability in physiological fluids in previous studies, little is understood about their structure and mechanical properties. Herein we report the surface and internal structures and mechanical properties of dendron OEGs, and quantitatively determine their ability to avoid non-specific adhesion of blood platelets. To gain insight into the interfacial force interactions, we measured the coarse-scale surface force acting on cell-sized particles and mapped the nanoscopic pinning centers by fast force mapping
Lensless Tomographic Imaging of Near Surface Structures of Frozen Hydrated Malaria-Infected Human Erythrocytes by Coherent X-Ray Diffraction Microscopy
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, âghost cellsâ were prepared by the removal of cytoplasm. From two-dimensional images, we found that the surface of erythrocytes after 32âh of infection became much rougher compared to that of healthy, uninfected erythrocytes. The Gaussian roughness of an infected erythrocyte surface (69ânm) is about two times larger than that of an uninfected one (31ânm), 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 64ânm for uninfected and 80ânm 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
Wnt/ÎČ-catenin signaling induces axial elasticity patterns of Hydra extracellular matrix
Summary: The extracellular matrix (ECM) plays crucial roles in animal development and diseases. Here, we report that Wnt/ÎČ-catenin signaling induces the ECM remodeling during Hydra axis formation. We determined the micro- and nanoscopic arrangement of fibrillar type I collagen along Hydraâs body axis using high-resolution microscopy and X-ray scattering. Elasticity mapping of the ECM ex vivo revealed distinctive elasticity patterns along the body axis. A proteomic analysis of the ECM showed that these elasticity patterns correlate with a gradient-like distribution of metalloproteases along the body axis. Activation of the Wnt/ÎČ-catenin pathway in wild-type and transgenic animals alters these patterns toward low ECM elasticity patterns. This suggests a mechanism whereby high protease activity under control of Wnt/ÎČ-catenin signaling causes remodeling and softening of the ECM. This Wnt-dependent spatiotemporal coordination of biochemical and biomechanical cues in ECM formation was likely a central evolutionary innovation for animal tissue morphogenesis
Influence of length and conformation of saccharide head groups on the mechanics of glycolipid membranes: Unraveled by off-specular neutron scattering.
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.peerReviewe
Three-Legged 2,2âČ-Bipyridine Monomer at the Air/Water Interface: Monolayer Structure and Reactions with Ni(II) Ions from the Subphase
The behavior of compound <b>2</b> [1,3,5-triÂ(2,2âČ-bipyridin-5-yl)Âbenzene]
with three bipyridine units arranged in a star geometry is investigated
in the presence and absence of NiÂ(ClO<sub>4</sub>)<sub>2</sub>. Its
properties at the airâwater 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 <b>2</b> within the monolayers formed stays
almost vertical at the interface and that at high Ni<sup>2+</sup>/<b>2</b> (Ni<sup>2+</sup>/<b>2</b> = 4000, 20âČ000) ratios
two of the three bipyridine units of <b>2</b> are complexed,
resulting in supramolecular sheets that are likely composed of arrays
of linear metalâorganic complexation polymers