The thermotropic behavior of multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC), or of DPPC in admixture with cardiolipin or cholesterol, in the presence of various N-alkyl derivatives of both adriamycin-14-valerate has been investigated by high sensitivity differential scanning calorimetry. The analogues, particularly the 14-valerate derivatives, which were most lipophilic as judged by their lipid/buffer, and to a lesser extent by their octanol/buffer, partition coefficients, were the most effective in depressing the tm of the investigated lipids; correlations, however, were not absolute. Other factors, such as the distribution of the drugs between the solid and liquid-crystalline phases of the bilayer, were also important to the observed membrane perturbations. With all anthracyclines, however, no major changes in the transition enthalpy were observed. In the case of vesicles prepared from pure DPPC, curve fitting analysis based on ideal solution theory (J.M. Sturtevant (1984) Proc. Natl. Acad. Sci. USA 81, 1398-1400) applied at relatively low drug concentrations where single peak transitions were produced, adequately described the differential scanning calorimetric results. At high drug concentrations, however, the presence of multipeak transitions were indicative of non-ideality.

No description available.

Diss. (sammanfattning) Umeå : Univ., 2009. / Härtill 4 uppsatser.

No description available.

Tumour necrosis factor-stimulated gene-6 (TSG-6) is a protein expressed in a wide range of cell types and tissues, predominantly in response to inflammatory stimuli. The expression of TSG-6 is believed to be associated with the protection of tissues from the damaging effects of inflammation. In animal models treatment with TSG-6 protein has been found to reduce inflammatory damage in myocardial infarction, corneal injury and arthritis. Endogenous TSG-6 production has been suggested to play a protective role in inflammatory arthritis and has been implicated in bone homeostasis. The expression of TSG-6 is also essential in the process of cumulus matrix formation that occurs around the oocyte in the periovulatory period and is necessary for successful ovulation and fertilisation. In many cases the mechanism underlying a particular TSG-6 function is not fully understood. TSG-6 has numerous binding partners including the serum glycoprotein inter-alpha-inhibitor (IαI), the growth factor bone morphogenetic protein-2 (BMP-2) and the extracellular matrix protein fibronectin, as well as glycosaminoglycans (GAGs) such as hyaluronan and heparan sulphate (HS). The TSG-6 protein is mostly composed of contiguous Link and CUB domains, with the majority of ligand binding sites identified within the Link module. The CUB domain of TSG-6 has been less extensively studied. Here biophysical techniques have been used to investigate the TSG-6 interactome including both the Link module and CUB domain. Intrinsic fluorescence spectroscopy was used to establish the metal-ion binding properties of the CUB domain, which was established to have a high affinity Ca2+-binding site. Using surface plasmon resonance (SPR), a novel metal-ion dependent interaction was found for the CUB domain of TSG-6 and the heavy chains (HCs) of IαI. Investigation using mutants of both the CUB domain of TSG-6 and HC of IαI established that the metal-ion binding sites within each protein are involved in the interaction. SPR analysis was also used to define the affinities and binding sites for TSG-6 interactions with fibronectin and BMP-2. High affinity interactions between TSG-6 ligands were also revealed (e.g. BMP-2 and HC, fibronectin and HC) and their binding sites defined. The discovery of the novel interactions between these TSG-6 ligands suggests crosstalk within the TSG-6 interactome, with the potential for ternary complex formation or indeed hierarchical orders of binding. Thus work was undertaken to develop a passivated lipid bilayer platform for use with surface sensitive techniques. This platform was used to investigate the hierarchy of protein and GAG interactions using quartz crystal microbalance with dissipation monitoring (QCM-D) and dual polarisation interferometry (DPI). The investigation revealed a novel role for the Link module of TSG-6 in heparin condensation, potentially via protein dimerisation and/or oligomerisation which could affect heparin/HS functions within the extracellular matrix (ECM). Thus the biophysical analysis of TSG-6 presented here has identified novel interactions and functions of TSG-6 which may provide mechanisms for the protective functioning of TSG-6 in inflammation and its ECM structuring role in ovulation.

Lipid bilayers and lipid-associated proteins play crucial roles in biology. As in vivo studies and manipulation are inherently difficult, membrane-mimetic systems are useful for the investigation of lipidic phases, lipid–protein interactions, membrane protein function and membrane structure in vitro. This dissertation describes a route to leverage the programmability of DNA nanotechnology to create DNA-encircled bilayers (DEBs), a novel nano-scaled membrane-mimetic system. DEBs are made of multiple copies of an alkylated oligonucleotide hybridized to a single-stranded minicircle, in which up to two alkyl chains per helical turn point to the inside of the toroidal DNA ring. When phospholipids are added, a bilayer is observed to self-assemble within the ring such that the alkyl chains of the oligonucleotides stabilize the hydrophobic rim of the bilayer to prevent formation of vesicles and support thermotropic lipid phase transitions. This straight-forward and robust route enables the rational design of DEBs so that their size, shape or functionalization can be adapted to the specific needs of biophysical investigations of lipidic phases and the properties of membrane proteins. Next, we optimized the DEB system to provide proper anchoring of a large variety of lipids by creating an improved DNA scaffold. This scaffold, called DNA double-decker, consists of two interconnected DNA minicircles stacked on top of each other. In comparison to the DNA minicircle in DEB system, this scaffold is two times thicker and contains two times more hydrophobic strands, which should increase the stability of the lipid bilayer rim. Finally, we explored the option of using DEBs in studies of GPCRs using CCR5 as a model protein. The CCR5 was labeled with DNA strands, purified and characterized. The strands on CCR5 are complementary to the protruding strands on the DNA minicircle in DEBs. This can allow the reconstitution of GPCRs inside DEBs with controlled orientation of the receptor.

The biological membrane, which is composed of a lipid bilayer embedded with numerous proteins, defines the cell boundary, separating the cell interior from the external environment. It serves as a gatekeeper and entry point for various molecular and ionic species. This thesis describes experimental and simulation studies of the interactions of carbon nanotubes (CNTs) with model membranes (lipid bilayers). The unique properties of CNTs make them ideal candidates for many nanotechnological applications. They can, however, pose a potential risk as toxins. While research into the positive benefits of CNTs continues, very little is known about their basic interactions with cellular components. It is particularly important to understand the interaction of CNTs with biological membranes, which form the primary physical barrier surrounding a cell. Coarse grained molecular dynamics (MD) simulations and atomic force microscopy (AFM) have been used to study the interactions of CNTs and lipid bilayers. They are investigated in a controlled manner using MD simulations, while AFM has allowed the controlled approach-to-contact and insertion of CNTs into bilayers. A number of effects are reported, including lipid creep along the CNT and bilayer thickening upon contact. The robustness of this response is established using different force fields and lipid species. The experimental results show an unusual reaction to mechanical indentation, and are further backed by MD simulations. The lipid bilayer response to multiple CNTs is studied and the effects of CNTs on bilayer conformation and lipid diffusion are reported. CNT internalisation from the solvent is observed in the simulations. Indeed, many of the observed phenomena are reminiscent of those known from the field of membrane protein. This project focuses on understanding the basic molecular interactions of CNTs with lipid bilayers and addresses the gap between experimental and computational work.

This thesis focuses on the use of biomolecular assemblies for creating materials with novel properties. Several aspects of biomimetic materials have been investigated, from fundamental studies on membrane shaping molecules to the integration of biomolecules with inorganic materials. Triply periodic minimal surfaces (TPMS) are mathematically defined surfaces that partition space and present a large surface area in a confined space. These surfaces have analogues in many physical systems. The endoplasmic reticulum (ER) can form intricate structures and it acts as a replica for the wing scales of the butterfly C. rubi, which is characterized by electron microscopy and reflectometry. It was shown to contain a photonic crystal and an analogue to a TPMS. These photonic crystals have been replicated in silica and titania, leading to blue scales with replication on the nanometer scale. Replicas analyzed with left and right handed polarized light are shown be optically active. A macroporous hollow core particle was synthesized using a double templating method where a swollen block copolymer was utilized to create polyhedral nanofoam. Emulsified oil was used as a secondary template which gave hollow spheres with thin porous walls. The resulting material had a high porosity and low thermal conductivity. The areas of inorganic materials and functional biomolecules were combined to create a functional nanoporous endoskeleton. The membrane protein ATP synthase were incorporated in liposomes which were deposited on nanoporous silica spheres creating a tight and functional membrane. Using confocal microscopy, it was possible to follow the transport of Na+ through the membrane. Yop1p is a membrane protein responsible for shaping the ER. The protein was purified and reconstituted into liposomes of three different sizes. The vesicles in the 10-20 nm size range resulted in tubular structures. Thus, it was shown that Yop1p acts as a stabilizer of high curvature structures. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Submitted. Paper 4: Submitted. Paper 5: Submitted.</p>

Das stäbchenförmige Bakterium Escherichia coli teilt sich in zwei gleich große Tochterzellen. Dies ist nur möglich, wenn sich die Zelle in der Mitte teilt. Bei E. coli wird die Zellteilung durch den Zusammenschluss der FtsZ-Proteine an der Membran zum Z-Ring eingeleitet. Topologische Regulierung des Z-Ringes erfolgt durch räumlich-zeitliche Oszillationen von Min-Proteinen zwischen den beiden Zellpolen. MinC, MinD und MinE binden an und lösen sich von der Membran unter Hydrolyse von ATP und in antagonistischer Art und Weise, was zu einer alternierenden Ansammlung von MinC und MinD an den Zellpolen führt. Gemittelt über die Zeit ergibt sich somit ein MinD-Verteilungsprofil, das maximale Konzentration an den Zellpolen und ein Minimum in der Zellmitte aufweist. MinC bindet an MinD und folgt somit seiner Verteilung. Der Zusammenschluss von FtsZ-Proteinen wird durch MinC unterbunden, und somit kann sich der Z-ring nur an einer Position herausbilden, die ein Minimum an MinC aufweist - der Zellmitte. Das Min-system wurde in der Vergangenheit auch mit einem in-vitro-Ansatz untersucht, indem Min-Proteine in künstliche, aufliegende Lipiddoppelschichten (supported lipid bilayers, SLB) rekonstitutiert wurden. Dabei bildeten die Min-Proteine kein oszillierendes Muster aus, sondern organisierten sich vielmehr in parallelen und propagierenden Wellen (Loose, 2008, Science, 320). In diesen in-vitro-Experimenten war das Membransubstrat wesentlich größer als die Wellenlänge der Min-Proteinwellen. In vivo hingegen ist die Länge der Zelle in der gleichen Größenordnung wie die charakteristische Länge des Oszillationsmusters der Min-Proteine. Daher war es das Ziel dieser Arbeit, den Einfluß einer beschränkten Fläche und geometrischer Formgebung der künstlichen Lipiddoppelschichten auf die Wellenpropagation der Min-Protein zu untersuchen. Flächige Beschränkung künstlicher Membranen erfolgte durch Mikrostrukturtechnologie. Deckglässchen wurden mit einer Goldschicht und mikroskopischen Aussparungen unterschiedlicher geometrischer Formen strukturiert. Funktionale SLBs bildeten sich nur auf Glasflächen ohne Goldbeschichtung aus. Nach der Rekonstitution der Min-Proteine, organisierten sich diese auf den Membranstücken in parallele Wellen. Dabei bestimmte die flächige Beschränkung der künstlichen Membranen die Ausbreitungsrichtung der Min-Proteinwellen. Min-Proteinwellen konnten entlang gekrümmter Membranstreifen, in Ring- und sogar in Slalomstrukturen geleitet werden. In geraden, länglichen Strukturen richteten sich die Wellen entlang der längsten Achse aus. Kopplung von Proteinwellen auf räumlich getrennten Membranstücken in Abhängigkeit des Abstandes und des sogenannten Molecular Crowdings in der wässrigen Lösung konnte ebenfalls beobachtet werden. Diese Kopplung ist ein Indiz für inhomogene Proteinverteilungen in der Lösung oberhalb der Membran. Desweiteren konnten Min-Proteinwellen auch in diversen dreidimensionalen künstlichen Membranen rekonstitituiert werden. Im Wildtyp von E. coli ähneln die Min-Proteindynamiken der einer Oszillation mit einer charakteristischen Länge von 5 µm. Auf SLBs, bilden Min-Proteine Wellen mit einer Wellenlänge aus, die ca. zehnmal größer ist als in vivo. Dieser Unterschied zwischen der in-vivo- und der in-vitro-Welt wurde untersucht und diskutiert. In vitro konnte die Wellenlänge um 50 % durch Erhöhung des Molecular Crowding in der Lösung sowie um 33 % durch Temperaturerhöhung verkleinert werden. Das oszillierende Muster könnte dahingegen eine Folge der Kompartimentierung sein. Erste Versuche, das Min-System in geschlossene Membrankompartimente zu rekonstitutieren, wurden getestet. / Escherichia coli, a rod-like bacterium, divides by binary fission. Cell division into two daughter cells of equal size requires that fission takes place at a midcell position. In E. coli, cell division is initiated by assembly of the FtsZ-proteins at the inner membrane to the Z-ring. Topological regulation of the Z-ring is achieved by spatiotemporal pole-to-pole oscillations of Min-proteins. MinC, MinD and MinE bind to and detach from - under hydrolysis of ATP - the membrane in an antagonistic manner leading to an alternating accumulation of MinC and MinD at the cell poles. Averaged over time, the distribution profile of MinD exhibits maximal concentration at the cell poles and a minimum at the cell center. MinC binds to MinD and thus follows its distribution. FtsZ assembly is inhibited by MinC and therefore the Z-ring can only form at a cell position low in MinC - at the cell center. In the past, the Min-system was also investigated in an in vitro approach by reconstitution of Min-proteins into a supported lipid bilayer (SLB). Here, Min-proteins did not self-organize into an oscillatory pattern but into parallel and propagating waves (Loose, 2008, Science, 320). In this in vitro assay, the membrane substrate was infinitely large compared to the wavelength. However, in vivo, the cell length is on the same order of magnitude as the respective length scale of the oscillatory pattern of Min-proteins. Therefore, we wished to investigate the effect of lateral confinement and geometric structuring of artificial lipid bilayers on the Min-protein wave propagation. Lateral confinement of artificial membranes was achieved by microfabrication technology. Glass slides were patterned by a gold coating with microscopic windows of different geometries, and functional SLBs were only formed on uncoated areas. Upon reconstitution, Min-proteins organized into parallel waves on the geometric membrane patches. Confinement of the artificial membranes determined the direction of propagation of Min-protein waves. Min-protein waves could be guided along curved membrane stripes, in rings and even along slalom-geometries. In elongated membrane structures, the protein waves always propagate along the longest axis. Coupling of protein waves across spatially separated membrane patches was observed, dependent on gap size and level of molecular crowding of the aqueous media above the bilayer. This indicates the existence of an inhomogeneous and dynamic protein gradient in the solution above the membrane. Furthermore, reconstitution of Min-protein waves in various three-dimensional artificial membranes was achieved. In wild-type E. coli, Min-protein dynamics resemble that of an oscillation with a characteristic length scale of 5 µm. On supported lipid bilayers, Min-proteins self-organize into waves with a wavelength approximately 10-fold larger than in vivo. These discrepancies between the in vivo and in vitro world were investigated and discussed. In vitro, the wavelength could be decreased by a factor of 50 % by increase of the molecular crowding in solution and by 33 % through temperature increase. The oscillatory pattern is thought to be a consequence of compartmentalization and first attempts to encapsulate the Min-system in closed bilayer compartments are presented.

Imaging mass spectrometry (MS) analysis allows scientists the ability to obtain spatial and chemical information of analytes on a wide variety of surfaces. The ability to image biological analytes is an important tool in many areas of life science research, including: the ability to map pharmaceutical drugs in targeted tissue, to spatially determine the expression profile of specific proteins in healthy vs. diseased tissue states, and to rapidly interrogate biomolecular microarrays. However, there are several avenues for improving the imaging MS experiment for biological samples. Three significant directions this work addresses include: (1) reducing chemical noise and increasing analyte identification by developing sample preparation methodologies, (2) improving the analytical figures of merit (i.e., spatial resolution, analysis time) by implementing a spatially dynamic optical system, and (3) increasing both mass spectral resolution and ion detection sensitivity by modifying a commercial time-of-flight (TOF) MS. Firstly, sample methodology schemes presented in these studies consist of obtaining both ?top-down? and ?bottom-up? information. In that, both intact mass and peptide mass fingerprinting data can be obtained to increase protein identification. This sample methodology was optimized on protein microarrays in preparation for bio tissue analysis. Other work consists of optimizing novel sample preparation strategies for hydrated solid-supported lipid bilayer studies. Sample methods incorporating nanomaterials for laser desorption/ionization illustrate the ability to perform selective ionization of specific analytes. Specifically, our results suggest that silver nanoparticles facilitate the selective ionization of olefin containing species (e.g., steroids, vitamins). Secondly, an advanced optical design incorporating a spatially dynamic optical scheme allows for laser beam expansion, homogenization, collimation, shaping, and imaging. This spatially dynamic optical system allows user defined beam shapes, decreases analysis times associated with mechanical movement of the sample stage, and is capable of increasing the MS limits of detection by simultaneously irradiating multiple spots. Lastly, new data acquisition strategies (multiple anode detection schemes) were incorporated into a commercial time-of-flight mass spectrometer to increase both sensitivity and resolution in a matrix assisted laser desorption/ionization mass spectrometer. The utility of this technique can be applied to many different samples, where high mass spectral resolution allows for increased mass measurement accuracy.