No description available.

No description available.

No description available.

Die Struktur der Plasmamembran ist von deren Lipid- und Proteinzusammensetzung abhängig und wird durch die Anbindung an das unterliegende Zytoskelett beeinflusst. Das Ziel der vorliegenden Arbeit war die Untersuchung eines neuen Modellsystems basierend auf po­ren­über­span­nen­den Membranen, welches sowohl die heterogene Lipidzusammensetzung als auch den Einfluss eines unterliegenden Netzwerks berücksichtigt. Lipidmembranen, zusammengesetzt aus der „raft“-ähnlichen Lipidmischung DOPC/Sphingo­myelin/Cho­les­terin (40:40:20), wurden auf porösen, hochgeordneten Siliziumsubstraten mit Po­ren­durch­messern von 0.8, 1.2 und 2.0 µm durch Spreiten und Fusion von Riesenvesikeln (giant unilamellar vesicles, GUVs) präpariert. Die mikroskopische Phasenseparation in koexistierenden flüssig-geordneten (liquid ordered, lo) und flüssig-ungeordneten (liquid disordered, ld) Domänen wurde stark durch das unterliegende poröse Substrat beeinflusst. Die Größe der lo-Domänen konnte durch die Porengröße des Siliziumsubstrats, die Temperatur und den Cholesteringehalt der Membran, welcher durch Zugabe von Methyl-β-Cyclodextrin moduliert wurde, kontrolliert werden. Die Bindung der Shiga Toxin B-Untereinheit (STxB) an po­ren­überspannende Membranen, dotiert mit 5 mol% des Rezeptorlipids Gb3, führte zu einem Anstieg des Anteils der lo-Phase. Außerdem wurde die Bildung von lo-Domänen in nicht-phasenseparierten Membranen, zusammengesetzt aus DOPC/Sphingomyelin/Cholesterin/Gb3 (65:10:20:5), durch die Shiga Toxin-Bindung induziert. Ein Anstieg des Anteils der lo-Phase konnte ebenfalls bei der Bindung der pentameren Cholera Toxin B-Untereinheit (CTxB) an po­ren­überspannende Membranen, dotiert mit 1 mol% des Rezeptorlipids GM1, beobachtet werden. Des Weiteren wurde der Einfluss der chemischen Struktur des Gb3-Moleküls auf die Shiga Toxin-Bindung und die Reorganisation von festkörperunterstützten Membranen (solid supported membranes, SSMs) untersucht. Die STxB-Bindung an α-hydroxyliertes Gb3 erhöhte signifikant den Anteil der lo-Phase, während eine cis-Doppelbindung zur Bildung einer weiteren lo-Phase führte, die vermutlich ungesättigte (Glyko-)Sphingolipide und Cholesterin enthält. Im Falles des ungesättigten Gb3 konnte außerdem eine Kondensation zu größeren Domänen nach der STxB-Bindung beobachtet werden. Die genaue Phasenzuordnung der eingesetzten Glykospingolipide vor der Proteinbindung ist bisher unbekannt. Daher wurde das Phasenverhalten eines fluoreszierenden Polyen-Ga­lac­to­ce­re­bro­sids untersucht, welches bevorzugt in der lo-Phase von GUVs angereichert war. Dieser neue, intrinsische Fluorophor vermag als Grundlage für weitere Studien zum Phasenverhalten von Glykosphingolipiden dienen.

Endocytosis is one of the most fundamental mechanisms by which the cell communicates with its surrounding. Specific signals are transduced through the cell membrane by a complex interplay between proteins and lipids. Clathrin depended endocytosis is one of important signalling pathways which leads to budding of the plasmalemma and a formation of endosomes. The FCHo2 is an essential protein at the initial stage of the this process. In is a membrane binding protein containing BAR (BIN, Amphiphysin, Rvs) domain which is responsible for a membrane binding. Although numerous valuable work on BAR proteins was published recently, the mechanistic description of a BAR domain functionality is missing. In present work we applied in vitro systems in order to gain knowledge about molecular basis of the activity of the FCHo2 BAR domain. In our studies we used supported lipid bilayers (SLBs) and lipid monolayers as s model membrane system. The experiments were carried out with a minimal number of components including the purified FCHo2 BAR domain. Using SLBs we showed that the BAR domain can bind to entirely flat bilayers. We also demonstrated that these interactions depend on the negatively charged lipid species incorporated in the membrane. We designed an assay which allows to quantify the membrane tubulation. We found out that the interaction of the FCHo2 BAR domain with the lipid membrane is concentration dependent. We showed that an area of the bilayer deformed by the protein depends on the amount of the used BAR domain. In order to study the relation between the mobility of lipids and the activity of FCHo2 BAR domain we designed a small-volume monolayer trough. The design of this micro-chamber allows for the implementation of the light microscopy. We demonstrated that the measured lipid diffusion in the monolayer by our new approach is in agreement with literature data. We carried out fluorescence correlation spectroscopy (FCS) experiments at different density of lipids at the water-air interface.We showed that the FCHo2 BAR domain binding affinity is proportional to the mean molecular area (MMA). We additionally demonstrated that the increased protein binding is correlated with the higher lipid mobility in the monolayer. Additionally, by curing out high-speed atomic force microscopy (hsAFM) we acquired the structural information about FCHo2 BAR domains orientation at the membrane with a high spatio-temporal resolution. Obtained data indicate the BAR domains interact witheach other by many different contact sites what results in a variety of protein orientations in a protein assemble.

No description available.

Recent experimental studies indicate that polymeric structures with a well-adjusted balance of amphiphilic parts may translocate through self-assembled phospholipid bilayers and enhance the passive trans-membrane transport of smaller molecules. Using a coarse grained lattice Monte Carlo model with explicit solvent we investigate self-assembled lipid bilayers interacting with a linear polymer chain under variation of the hydrophobicity of the chain. Here, we focus on the relationship between the chain's hydrophobicity and its translocation behavior through the membrane as well as induced membrane perturbations. We show, that there is an adsorption transition of the polymer at the bilayer interface, where effectively the solvent phase and the tail phase of the bilayer are equally repulsive for the polymer. Close to this adsorption threshold of the polymer both the translocation probability of the polymer as well as the permeability of the membrane with respect to solvent are enhanced significantly. The frequency of polymer translocation events can be understood quantitatively assuming a simple diffusion along a one-dimensional free energy profile, which is controlled by the effective lipophilicity of the chain and the tail-packing in the bilayer's core. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The coiled-coil motif is present in proteins from all kingdoms of life. Its structure is based on a repeating sequence of 7 amino acids with hydrophobic residues at positions 1 and 4, which folds into an alpha-helix. Two, or more, alpha-helices wind around each other based on hydrophobic interactions forming the coiled-coil. Structural variations include length, deviations from the canonical form based on the heptad repeat, as well as the orientation and number of alpha-helices. They are involved in a wide variety of cellular processes including vesicle tethering and signal transmission along their length. In order to transmit signal, the protein must be able to dynamically rearrange its structure. An outstanding example of a coiled-coil that needs to rearrange its structure to perform its function is the early endosomal tether EEA1, which has been shown to increase its flexibility upon binding to the active form of the small GTPase Rab5. That conformational change generates an entropic collapse that brings the ends of the protein closer to each other. Nevertheless, the recycling from the more flexible state to its original extended conformation was not addressed. Herein, the entropic collapse mechanism was further studied and the full EEA1 cycle between extended and flexible states described. In addition to these studies, other coiled-coil proteins were assessed to determine if they also experience a binding-induced entropic collapse. One of the strategies to investigate the entropic collapse mechanism was to compare the adhesive forces along the two alpha-helices of the EEA1 dimer in its extended and flexible conformations. To this end, an experiment was designed to unwind the dimer using optical tweezers, a force-spectroscopy method that uses a highly focused laser beam to manipulate microscopic objects. Each EEA1 monomer was attached to a distinct DNA piece using a site-specific enzymatic reaction. The DNA pieces were linked to two optically trapped micron-sized beads. And the distance between the optical traps increased to unwind the EEA1. A second strategy to investigate the entropic collapse was to evaluate EEA1 dynamics in solution using dual color fluorescence cross-correlation spectroscopy (dcFCCS). EEA1 C-termini was labeled with two different fluorophores. Fluctuations on fluorescent intensities caused by the dyes crossing a confocal volume were recorded over time. Based on an analysis of these fluctuations, a conformational change in EEA1 from semi-flexible to flexible upon addition of active Rab5 was described. This is in agreement with the previously reported entropic collapse. More importantly, EEA1 was shown to cycle between semi-flexible and flexible states by adding Rab5:GTP and waiting for the GTP to hydrolyse. To determine whether other proteins experience a binding-induced entropic collapse, coiled-coil proteins that share structural and functional similarities with EEA1 were evaluated. Rotary shadowing EM images of the target protein alone and binding with its suspected allosteric effector were compared. It was found that ELKS, a coiled-coil protein involved in vesicle trafficking, undergoes an increase in flexibility upon binding with the active form of Rab6. Thus, hinting that the entropic collapse may indeed be a general mode of action for at least a sub-group of long coiled-coil proteins. Overall, the major contributions of this thesis are to describe the full entropic collapse cycle on EEA1 and to show a second example of a coiled-coil protein experiencing a binding induced flexibility increase.:List of Figures List of Tables List of Equations List of Abbreviations 1 Introduction 1.1 EEA1 as an endosomal tether 2 Materials and Methods 2.1 Materials 2.2 Methods 2.2.1 Sub-cloning 2.2.2 Protein expression and purification 2.2.3 Protein-protein binding assays 2.2.4 Electron microscopy 2.2.5 Analysis of electron microscopy 2.2.6 Generation of DNA handles for protein-DNA conjugates 2.2.7 Adding SortaseA recognition site to EEA1 2.2.8 Protein-DNA conjugation3 2.2.9 Sample preparation for optical tweezers 2.2.10 Dual color labeling of EEA1 2.2.11 Fluorescence cross-correlation spectroscopy 2.2.12 Generation of dsDNA for dcFCCS calibration 2.2.13 RabGTPase nucleotide loading 2.2.14 Liposome preparation 2.2.15 MCBs preparation 3 Unwinding EEA1 coiled-coil domain 3.1 Introduction 3.1.1 Optical tweezers for EEA1 unwinding 3.1.2 SortaseA-catalysed ligation 3.2 Aims 3.3 Results 3.3.1 Optimization of SortaseA-catalysed ligation 3.3.2 Formation of EEA1-DNA handle conjugate 3.3.3 EEA1 unwinding experiments 3.4 Discussion 4 EEA1 entropic collapse is recyclable 4.1 Introduction 4.1.1 Advantages of dcFCCS vs FCS 4.1.2 Requirements for dcFCCS measurements 4.1.3 dcFCCS for end polymer dynamics analysis 4.2 Aims 4.3 Results 4.3.1 System preparation and dcFCCS calibration 4.3.2 Labelling of EEA1 4.3.3 Comparing FCS vs dcFCCS 4.3.4 EEA1 entropic collapse shown by dcFCCS 4.3.5 EEA1 flexibility change is recyclable 4.4 Discussion 5 Entropic collapse as a general mechanism 5.1 Introduction 5.2 Aims 5.3 Results 5.3.1 ELKS increases its flexibility upon binding active Rab6 5.3.2 p115-GM130 complex observed by rotary shadowing EM 5.4 Discussion 6 Conclusions and outlook References

Membranes separate uid compartments and can comprise transport structures for selective permeation. In biology, channel proteins are specialized in their atomic structure to allow transport of specific compounds (selectivity). Conformational changes in protein structure allow the control of the permeation abilities by outer stimuli (gating). In polymeric membranes, the selectivity is due to electrostatic or size-exclusion. It can thus be controlled by size variation or electric charges. Controllable permeation can be useful to determine particle-size distributions in continuous ow, e.g. in micro uidics and biomedicine to gain cell diameter profiles in blood. The present approach uses patterned polyethylene terephthalate (PET) membranes with hydrogel surface coating for permeation control by size-exclusion. The thermosensitive hydrogel poly(N-isopropylacrylamide) (PNIPAAm) is structured with a cross-shaped pore geometry. A change in the temperature of the water ow through the membrane leads to a pore shape variation. The temperature dependent behavior of PNIPAAm can be numerically modeled with a temperature expansion model, where the swelling and deswelling is depicted by temperature dependent expansion coefficients. In the present study, the free swelling behavior was implemented to the Finite Element tool ABAQUS for the complex composite structure of the permeation control membrane. Experimental values of the geometry characteristics were derived from microscopy images with the tool ImageJ and compared to simulation results. Numerical simulations using the derived thermomechanical model for different pore geometries (circular, rectangle, cross and triangle) were performed. With this study, we show that the temperature expansion model with values from the free swelling behavior can be used to adequately predict the deformation behavior of the complex membrane system. The predictions can be used to optimize the behavior of the membrane pores and the overall performance of the smart membrane.

Endocytosis is one of the most fundamental mechanisms by which the cell communicates with its surrounding. Specific signals are transduced through the cell membrane by a complex interplay between proteins and lipids. Clathrin depended endocytosis is one of important signalling pathways which leads to budding of the plasmalemma and a formation of endosomes. The FCHo2 is an essential protein at the initial stage of the this process. In is a membrane binding protein containing BAR (BIN, Amphiphysin, Rvs) domain which is responsible for a membrane binding. Although numerous valuable work on BAR proteins was published recently, the mechanistic description of a BAR domain functionality is missing. In present work we applied in vitro systems in order to gain knowledge about molecular basis of the activity of the FCHo2 BAR domain. In our studies we used supported lipid bilayers (SLBs) and lipid monolayers as s model membrane system. The experiments were carried out with a minimal number of components including the purified FCHo2 BAR domain. Using SLBs we showed that the BAR domain can bind to entirely flat bilayers. We also demonstrated that these interactions depend on the negatively charged lipid species incorporated in the membrane. We designed an assay which allows to quantify the membrane tubulation. We found out that the interaction of the FCHo2 BAR domain with the lipid membrane is concentration dependent. We showed that an area of the bilayer deformed by the protein depends on the amount of the used BAR domain. In order to study the relation between the mobility of lipids and the activity of FCHo2 BAR domain we designed a small-volume monolayer trough. The design of this micro-chamber allows for the implementation of the light microscopy. We demonstrated that the measured lipid diffusion in the monolayer by our new approach is in agreement with literature data. We carried out fluorescence correlation spectroscopy (FCS) experiments at different density of lipids at the water-air interface.We showed that the FCHo2 BAR domain binding affinity is proportional to the mean molecular area (MMA). We additionally demonstrated that the increased protein binding is correlated with the higher lipid mobility in the monolayer. Additionally, by curing out high-speed atomic force microscopy (hsAFM) we acquired the structural information about FCHo2 BAR domains orientation at the membrane with a high spatio-temporal resolution. Obtained data indicate the BAR domains interact witheach other by many different contact sites what results in a variety of protein orientations in a protein assemble.