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Institute
Giant unilamellar vesicles are an important tool in todays experimental efforts to understand the structure and behaviour of biological cells. Their simple structure allows the isolation of the physical elastic properties of the lipid membrane. A central physical
property is the bending energy of the membrane, since the many different shapes of giant vesicles can be obtained by finding the minimum of the bending energy. In the spontaneous curvature model the bending energy is a function of the bending rigidity as well as the mean curvature and an additional parameter called the spontaneous curvature, which describes an internal preference of the lipid-bilayer to bend towards one side or the other. The spontaneous and mean curvature are local properties of the membrane.
Additional constraints arise from the conservation of the membrane surface area and the enclosed volume, which are global properties.
In this thesis the spontaneous curvature model is used to explain the experimental observation of a periodic shape oscillation of a giant unilamellar vesicle that was filled with a protein complex that periodically binds to and unbinds from the membrane.
By assuming that the binding of the proteins to the membrane induces a change in the spontaneous curvature the experimentally observed shapes could successfully be explained. This involves the numerical solution of the differential equations as obtained from the minimization of the bending energy respecting the area and volume constraints, the so called shape equations. Vice versa this approach can be used to estimate the spontaneous curvature from experimentally measurable quantities.
The second topic of this thesis is the analysis of concentration gradients in rigid conic membrane compartments. Gradients of an ideal gas due to gravity and gradients generated by the directed stochastic movement of molecular motors along a microtubulus were considered. It was possible to calculate the free energy and the bending energy analytically for the ideal gas. In the case of the non-equilibrium system with molecular motors, the characteristic length of the density profile, the jam-length, and its dependency on the opening angle of the conic compartment have been calculated in the mean-field limit.
The mean field results agree qualitatively with stochastic particle simulations.
Life on Earth is diverse and ranges from unicellular organisms to multicellular creatures like humans. Although there are theories about how these organisms might have evolved, we understand little about how ‘life’ started from molecules. Bottom-up synthetic biology aims to create minimal cells by combining different modules, such as compartmentalization, growth, division, and cellular communication.
All living cells have a membrane that separates them from the surrounding aqueous medium and helps to protect them. In addition, all eukaryotic cells have organelles that are enclosed by intracellular membranes. Each cellular membrane is primarily made of a lipid bilayer with membrane proteins. Lipids are amphiphilic molecules that assemble into molecular bilayers consisting of two leaflets. The hydrophobic chains of the lipids in the two leaflets face each other, and their hydrophilic headgroups face the aqueous surroundings. Giant unilamellar vesicles (GUVs) are model membrane systems that form large compartments with a size of many micrometers and enclosed by a single lipid bilayer. The size of GUVs is comparable to the size of cells, making them good membrane models which can be studied using an optical microscope. However, after the initial preparation, GUV membranes lack membrane proteins which have to be reconstituted into these membranes by subsequent preparation steps. Depending on the protein, it can be either attached via anchor lipids to one of the membrane leaflets or inserted into the lipid bilayer via its transmembrane domains.
The first step is to prepare the GUVs and then expose them to an exterior solution with proteins. Various protocols have been developed for the initial preparation of GUVs. For the second step, the GUVs can be exposed to a bulk solution of protein or can be trapped in a microfluidic device and then supplied with the protein solution. To minimize the amount of solution and for more precise measurements, I have designed a microfluidic device that has a main channel, and several dead-end side channels that are perpendicular to the main channel. The GUVs are trapped in the dead-end channels. This design exchanges the solution around the GUVs via diffusion from the main channel, thus shielding the GUVs from the flow within the main channel. This device has a small volume of just 2.5 μL, can be used without a pump and can be combined with a confocal microscope, enabling uninterrupted imaging of the GUVs during the experiments. I used this device for most of the experiments on GUVs that are discussed in this thesis.
In the first project of the thesis, a lipid mixture doped with an anchor lipid was used that can bind to a histidine chain (referred to as His-tag(ged) or 6H) via the metal cation Ni2+. This method is widely used for the biofunctionalization of GUVs by attaching proteins without a transmembrane domain. Fluorescently labeled His-tags which are bound to a membrane can be observed in a confocal microscope. Using the same lipid mixture, I prepared the GUVs with different protocols and investigated the membrane composition of the resulting GUVs by evaluating the amount of fluorescently labeled His-tagged molecules bound to their membranes. I used the microfluidic device described above to expose the outer leaflet of the vesicle to a constant concentration of the His-tagged molecules. Two fluorescent molecules with a His-tag were studied and compared: green fluorescent protein (6H-GFP) and fluorescein isothiocyanate (6H-FITC). Although the quantum yield in solution is similar for both molecules, the brightness of the membrane-bound 6H-GFP is higher than the brightness of the membrane-bound 6H-FITC. The observed difference in the brightness reveals that the fluorescence of the 6H-FITC is quenched by the anchor lipid via the Ni2+ ion. Furthermore, my measurements also showed that the fluorescence intensity of the membranebound His-tagged molecules depends on microenvironmental factors such as pH. For both 6H-GFP and 6H-FITC, the interaction with the membrane is quantified by evaluating the equilibrium dissociation constant. The membrane fluorescence is measured as a function of the fluorophores’ molar concentration. Theoretical analysis of these data leads to the equilibrium dissociation constants of (37.5 ± 7.5) nM for 6H-GFP and (18.5 ± 3.7) nM for 6H-FITC.
The anchor lipid mentioned previously used the metal cation Ni2+ to mediate the bond between the anchor lipid and the His-tag. The Ni2+ ion can be replaced by other transition metal ions. Studies have shown that Co3+ forms the strongest bonds with the His-tags attached to proteins. In these studies, strong oxidizing agents were used to oxidize the Co2+ mediated complex with the His-tagged protein to a Co3+ mediated complex. This procedure puts the proteins at risk of being oxidized as well. In this thesis, the vesicles were first prepared with anchor lipids without any metal cation. The Co3+ was added to these anchor lipids and finally the His-tagged protein was added to the GUVs to form the Co3+ mediated bond. This system was also established using the microfluidic device.
The different preparation procedures of GUVs usually lead to vesicles with a spherical morphology. On the other hand, many cell organelles have a more complex architecture with a non spherical topology. One fascinating example is provided by the endoplasmic reticulum (ER) which is made of a continuous membrane and extends throughout the cell in the form of tubes and sheets. The tubes are connected by three-way junctions and form a tubular network of irregular polygons. The formation and maintenance of these reticular networks requires membrane proteins that hydrolyize guanosine triphosphate (GTP). One of these membrane proteins is atlastin. In this thesis, I reconstituted the atlastin protein in GUV membranes using detergent-assisted reconstitution protocols to insert the proteins directly into lipid bilayers.
This thesis focuses on protein reconstitution by binding His-tagged proteins to anchor lipids and by detergent-assisted insertion of proteins with transmembrane domains. It also provides the design of a microfluidic device that can be used in various experiments, one example is the evaluation of the equilibrium dissociation constant for membrane-protein interactions. The results of this thesis will help other researchers to understand the protocols for preparing GUVs, to reconstitute proteins in GUVs, and to perform experiments using the microfluidic device. This knowledge should be beneficial for the long-term goal of combining the different modules of synthetic biology to make a minimal cell.
Proteine sind an praktisch allen Prozessen in lebenden Zellen maßgeblich beteiligt. Auch in der Biotechnologie werden Proteine in vielfältiger Weise eingesetzt.
Ein Protein besteht aus einer Kette von Aminosäuren. Häufig lagern sich mehrere dieser Ketten zu größeren Strukturen und Funktionseinheiten, sogenannten Proteinkomplexen,
zusammen. Kürzlich wurde gezeigt, dass eine Proteinkomplexbildung bereits während der Biosynthese der Proteine (co-translational) stattfinden kann
und nicht stets erst danach (post-translational) erfolgt. Da Fehlassemblierungen von Proteinen zu Funktionsverlusten und adversen Effekten führen, ist eine präzise und verlässliche Proteinkomplexbildung sowohl für zelluläre Prozesse als auch für biotechnologische Anwendungen essenziell. Mit experimentellen Methoden lassen sich zwar u.a. die Stöchiometrie und die Struktur von Proteinkomplexen bestimmen,
jedoch bisher nicht die Dynamik der Komplexbildung auf unterschiedlichen Zeitskalen. Daher sind grundlegende Mechanismen der Proteinkomplexbildung noch nicht vollständig verstanden. Die hier vorgestellte, auf experimentellen Erkenntnissen aufbauende, computergestützte Modellierung der Proteinkomplexbildung erlaubt eine umfassende Analyse des Einflusses physikalisch-chemischer Parameter
auf den Assemblierungsprozess. Die Modelle bilden möglichst realistisch die experimentellen Systeme der Kooperationspartner (Bar-Ziv, Weizmann-Institut, Israel; Bukau und Kramer, Universität Heidelberg) ab, um damit die Assemblierung von Proteinkomplexen einerseits in einem quasi-zweidimensionalen synthetischen Expressionssystem (in vitro) und andererseits im Bakterium Escherichia coli (in vivo) untersuchen zu können. Mit Hilfe eines vereinfachten Expressionssystems, in dem die Proteine nur an die Chip-Oberfläche, aber nicht aneinander binden können, wird das theoretische Modell parametrisiert. In diesem vereinfachten in-vitro-System durchläuft die Effizienz der Komplexbildung drei Regime – ein bindedominiertes Regime, ein Mischregime und ein produktionsdominiertes Regime. Ihr Maximum erreicht die Effizienz dabei kurz nach dem Übergang vom bindedominierten ins Mischregime und fällt anschließend monoton ab. Sowohl im nicht-vereinfachten in-vitro- als auch im in-vivo-System koexistieren je zwei konkurrierende Assemblierungspfade: Im in-vitro-System erfolgt die Komplexbildung entweder spontan in wässriger Lösung (Lösungsassemblierung) oder aber in einer definierten Schrittfolge an der Chip-Oberfläche (Oberflächenassemblierung); Im in-vivo-System konkurrieren hingegen die co- und die post-translationale Komplexbildung. Es zeigt sich, dass die Dominanz der Assemblierungspfade im in-vitro-System zeitabhängig ist und u.a. durch die Limitierung und Stärke der Bindestellen auf der Chip-Oberfläche beeinflusst werden kann. Im in-vivo-System hat der räumliche Abstand zwischen den Syntheseorten der beiden Proteinkomponenten nur dann einen Einfluss auf die Komplexbildung, wenn die Untereinheiten schnell degradieren. In diesem Fall dominiert die co-translationale Assemblierung auch auf kurzen Zeitskalen deutlich, wohingegen es bei stabilen Untereinheiten zu einem Wechsel von der Dominanz der post- hin zu einer geringen Dominanz der co-translationalen Assemblierung kommt. Mit den in-silico-Modellen lässt sich neben der Dynamik u.a. auch die Lokalisierung der Komplexbildung und -bindung darstellen, was einen Vergleich der theoretischen Vorhersagen mit experimentellen Daten und somit eine Validierung der Modelle ermöglicht. Der hier präsentierte in-silico Ansatz ergänzt die experimentellen Methoden, und erlaubt so, deren Ergebnisse zu interpretieren und neue Erkenntnisse davon abzuleiten.
Glycosylphosphatidylinositols (GPIs) are highly complex glycolipids that serve as membrane anchors to a large variety of eukaryotic proteins. These are covalently attached to a group of peripheral proteins called GPI-anchored proteins (GPI-APs) through a post-translational modification in the endoplasmic reticulum. The GPI anchor is a unique structure composed of a glycan, with phospholipid tail at one end and a phosphoethanolamine linker at the other where the protein attaches. The glycan part of the GPI comprises a conserved pseudopentasaccharide core that could branch out to carry additional glycosyl or phosphoethanolamine units. GPI-APs are involved in a diverse range of cellular processes, few of which are signal transduction, protein trafficking, pathogenesis by protozoan parasites like the malaria- causing parasite Plasmodium falciparum. GPIs can also exist freely on the membrane surface without an attached protein such as those found in parasites like Toxoplasma gondii, the causative agent of Toxoplasmosis. These molecules are both structurally and functionally diverse, however, their structure-function relationship is still poorly understood. This is mainly because no clear picture exists regarding how the protein and the glycan arrange with respect to the lipid layer. Direct experimental evidence is rather scarce, due to which inconclusive pictures have emerged, especially regarding the orientation of GPIs and GPI-APs on membrane surfaces and the role of GPIs in membrane organization. It appears that computational modelling through molecular dynamics simulations would be a useful method to make progress. In this thesis, we attempt to explore characteristics of GPI anchors and GPI-APs embedded in lipid bilayers by constructing molecular models at two different resolutions – all-atom and coarse-grained.
First, we show how to construct a modular molecular model of GPIs and GPI-anchored proteins that can be readily extended to a broad variety of systems, addressing the micro-heterogeneity of GPIs. We do so by creating a hybrid link to which GPIs of diverse branching and lipid tails of varying saturation with their optimized force fields, GLYCAM06 and Lipid14 respectively, can be attached. Using microsecond simulations, we demonstrate that GPI prefers to “flop-down” on the membrane, thereby, strongly interacting with the lipid heads, over standing upright like a “lollipop”. Secondly, we extend the model of the GPI core to carry out a systematic study of the structural aspects of GPIs carrying different side chains (parasitic and human GPI variants) inserted in lipid bilayers. Our results demonstrate the importance of the side branch residues as these are the most accessible, and thereby, recognizable epitopes. This finding qualitatively agrees with experimental observations that highlight the role of the side branches in immunogenicity of GPIs and the specificity thereof. The overall flop-down orientation of the GPIs with respect to the bilayer surface presents the side chain residues to face the solvent. Upon attaching the green fluorescent protein (GFP) to the GPI, it is seen to lie in close proximity to the bilayer, interacting both with the lipid heads and glycan part of the GPI. However the orientation of GFP is sensitive to the type of GPI it is attached to. Finally, we construct a coarse-grained model of the GPI and GPI-anchored GFP using a modified version of the MARTINI force-field, using which the timescale is enhanced by at least an order of magnitude compared to the atomistic system.
This study provides a theoretical perspective on the conformational behavior of the GPI core and some of its branched variations in presence of lipid bilayers, as well as draws comparisons with experimental observations. Our modular atomistic model of GPI can be further employed to study GPIs of variable branching, and thereby, aid in designing future experiments especially in the area of vaccines and drug therapies. Our coarse-grained model can be used to study dynamic aspects of GPIs and GPI-APs w.r.t plasma membrane organization. Furthermore, the backmapping technique of converting coarse-grained trajectory back to the atomistic model would enable in-depth structural analysis with ample conformational sampling.