Biological membranes are mainly composed of two layers of lipids, various kinds of proteins and organic macromolecules, forming the protective barriers that separate the inner milieu of living cells from the environment. The possibility of penetrating the membrane is of great importance for biomedical applications. Recently, a lot of attention has been given to the mechanisms and the details of the interactions between the membrane and nanoparticles, as well as to the development of effective delivery strategies. A manipulation of the hydrophobicity of nanoparticles can facilitate the translocation through the membrane. Modifying the physical/chemical properties of the membrane through oxidation can also influence the delivery of nanoparticles or macromolecules into the cell. In this work, using coarse-grained molecular dynamics simulations, the passive translocation of nanoparticles with a size of about 1 nm and with tunable degrees of hydrophobicity through lipid membranes is studied. It is shown that a window of nanoparticle translocation with a sharp maximum is located at a certain hydrophobicity in between fully hydrophilic and fully hydrophobic characters. By combining direct simulations with umbrella sampling simulations, the free energy landscapes for nanoparticles covering a wide range of hydrophobicities are obtained. The directly observed translocation rate of the nanoparticles can be mapped to the mean escape rate through the calculated free energy landscapes, and the maximum of translocation can be related with the maximally flat free energy landscape. For nanoparticles with the balanced hydrophobicity, the bilayer forms a remaining barrier of a few kBT and can be spontaneously surmounted. Further investigations are conducted to explore the cooperative effects of a larger number of nanoparticles and their impact on membrane properties such as membrane permeability for solvent, the area per lipid, and the orientation order of lipid tails. By calculating the partition of nanoparticles between water and oil phases, the microscopic parameter, i.e. the hydrophobicity of nanoparticles, can be mapped to an experimentally accessible partition coefficient. The studies reveal a generic mechanism for spherical nanoparticles to overcome biological membrane-barriers without the need of biologically activated processes. Two oxidatively modified lipids are studied on coarse-grained level using molecular dynamics simulations. The findings support the view that lipid oxidation leads to a change of the lipid conformation: lipid tails tend to bend toward the lipid head-tail interface due to the presence of hydrophilic oxidized beads. This change in conformation can further influence structural properties, elasticity and membrane permeability: an increase of the area per lipid, accompanied with decrease of the membrane thickness and order parameter of the lipid tails; a sharp drop of stretching modulus; a significant increase of the membrane permeability for water. Oxidized lipid bilayers interacting with NPs of various degrees of hydrophobicity are further studied. The critical hydrophobicity corresponding to the maximum translocation rate of NPs, shifts towards the hydrophilic region, which coincides with the same decrease in percentage of the average hydrophobicity in the core of the membrane upon oxidation. Around the critical point of NPs' hydrophobicity, a significant increase of the translocation rate of NPs through the oxidized bilayers is observed, when compared to non-oxidized bilayers. This is associated with a deterioration of the free energy barrier for NPs inside the oxidized bilayers, resulting from oxidation effects. These findings are consistent with the studies of the mean escape rate through the free energy landscapes using Kramers theory. Regarding the membrane perturbation induced by NPs of various hydrophobicity, the data obtained with oxidized lipid bilayers present the same general trend as in the case of the non-oxidized lipid bilayer. These findings provide a better understanding of the interaction between NPs and oxidized lipid bilayers, and open a possibility to facilitate drug delivery.:1 Introduction 1 1.1 Lipid Bilayers 1 1.2 Oxidized Lipid Bilayers 2 1.3 Experimental Methodology 4 1.4 Lipid Models 5 1.5 The Lipid Bilayer Interacting with NPs 6 1.6 Thesis Overview 7 2 State of the art 9 2.1 Molecular Dynamics Simulations of Lipid Bilayers 9 2.1.1 Equations of Motion and the Integrations of Equations of Motion 10 2.1.2 Interaction Potentials 12 2.1.3 Periodic Boundary Conditions 14 2.1.4 Barostats and Thermostats 15 2.2 Umbrella Sampling Simulation 19 2.2.1 The Basics of Umbrella Sampling Method 20 2.2.2 Analyzing Umbrella Sampling Results by WHAM 23 2.2.3 The Principle of Choosing Bias Potential 24 3 Lipid Membranes interacting with Nanoparticles of Various Degrees of Hydrophobicity 25 3.1 Introduction 25 3.2 Coarse-grained Model and Simulation Setups 27 3.3 Results and Discussions 31 3.3.1 NPs-membrane Interactions 31 3.3.2 NPs Translocation 33 3.3.3 Concentration Effect of NPs 35 3.3.4 The Effect of Hydrophobicity on Kinetic Pathways 38 3.3.5 Potential of Mean Force 39 3.3.6 Hydrophobicity Scale 41 3.3.7 Solvent Permeation and Membrane Perturbation Induced by NPs 45 3.4 Summary 47 4 Coarse-grained Model of Oxidized Lipids and their Interactions with NPs of Varying Hydrophobicities 51 4.1 Introduction 51 4.2 Coarse-grained Model and Simulation Details 52 4.3 Results and Discussions 54 4.3.1 Characterizing the Oxidized Lipid Membranes 54 4.3.2 Oxidized Lipid Membranes Interacting with NPs of Various Degrees of Hydrophobicity 59 4.4 Summary 65 5 Summary and Outlook 69

Proteins are known to form functional clusters in plasma membranes. In order to identify individual proteins within clusters we developed a method to visualize by atomic force microscopy (AFM) the cytoplasmic surface of native plasma membrane, excised from Xenopus laevis oocyte and spread on poly-L-lysine coated glass. After removal of the vitelline membrane intact oocytes were brought in contact with coated glass and then rolled off. Inside-out oriented plasma membrane patches left at the glass surface were first identified with the lipid fluorescent marker FM1-43 and then scanned by AFM. Membrane patches exhibiting the typical phospholipid bilayer height of 5 nm showed multiple proteins, protruding from the inner surface of the membrane, with heights of 5 to 20 nm. Modelling plasma membrane proteins as spherical structures embedded in the lipid bilayer and protruding into the cytoplasm allowed an estimation of the respective molecular masses. Proteins ranged from 35 to 2,000 kDa with a peak value of 280 kDa. The most frequently found membrane protein structure (40/μm2) had a total height of 10 nm and an estimated molecular mass of 280 kDa. Membrane proteins were found firmly attached to the poly-L-lysine coated glass surface while the lipid bilayer was found highly mobile. We detected protein structures with distinguishable subunits of still unknown identity. Since X. laevis oocyte is a generally accepted expression system for foreign proteins, this method could turn out to be useful to structurally identify specific proteins in their native environment at the molecular level. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

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Proteins are known to form functional clusters in plasma membranes. In order to identify individual proteins within clusters we developed a method to visualize by atomic force microscopy (AFM) the cytoplasmic surface of native plasma membrane, excised from Xenopus laevis oocyte and spread on poly-L-lysine coated glass. After removal of the vitelline membrane intact oocytes were brought in contact with coated glass and then rolled off. Inside-out oriented plasma membrane patches left at the glass surface were first identified with the lipid fluorescent marker FM1-43 and then scanned by AFM. Membrane patches exhibiting the typical phospholipid bilayer height of 5 nm showed multiple proteins, protruding from the inner surface of the membrane, with heights of 5 to 20 nm. Modelling plasma membrane proteins as spherical structures embedded in the lipid bilayer and protruding into the cytoplasm allowed an estimation of the respective molecular masses. Proteins ranged from 35 to 2,000 kDa with a peak value of 280 kDa. The most frequently found membrane protein structure (40/μm2) had a total height of 10 nm and an estimated molecular mass of 280 kDa. Membrane proteins were found firmly attached to the poly-L-lysine coated glass surface while the lipid bilayer was found highly mobile. We detected protein structures with distinguishable subunits of still unknown identity. Since X. laevis oocyte is a generally accepted expression system for foreign proteins, this method could turn out to be useful to structurally identify specific proteins in their native environment at the molecular level. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Es ist eine allgemein akzeptierte Tatsache, dass der Kontakt von Proteinen mit synthetischen Materialien üblicherweise zur Proteinadsorption an der Materialoberfläche führt. Über den stattfindenden Prozess, insbesondere das Zusammenspiel zwischen Protein-Oberflächen-Wechselwirkungen und konformellen Änderungen der adsorbierten Proteine ist jedoch bisher nur wenig bekannt. In dieser Arbeit wird die ortsgerichtete Spinmarkierungstechnik (SDSL) auf die Strukturuntersuchung adsorbierter Proteine ausgeweitet. Diese nutzt das spezifische Einbringen einer spinmarkierte Seitenkette an gewünschte Positionen der Primärstruktur zur Analyse der Struktur und Dynamik diamagnetischer Proteine mittels der Elektronenspinresonanz(ESR)-Spektroskopie. Das globuläre Protein T4 Lysozym (T4L) wurde auf planare Modelloberflächen adsorbiert und strukturelle Änderungen in Abhängigkeit der physikalischen und chemischen Eigenschaften der Oberfläche verfolgt. Die spezifische Anbindung von T4L auf quarzgestützten zwitterionische Lipiddoppelschichten führt nur zu geringfügigen strukturellen Veränderungen des Proteins. Allerdings bildet sich eine makroskopisch geordnete Proteinschicht aus. Die Vorzugsrichtung der Proteine auf der Oberfläche kann durch Analyse der winkelabhängigen ESR-Spektren bestimmt werden. Die Wechselwirkung negativ geladener Oberflächen mit dem positiv geladenen T4L führt zu drastischeren Störungen der Proteinstruktur. Hierbei wird die Reaktion des Proteins auf den Kontakt mit einer fluiden quarzgestützten Lipiddoppelschicht, die das negativ geladenen Lipid Phosphatidylserin enthält, mit derer bei Adsorption auf einer ebenfalls negativ geladenen, jedoch rigiden Quarzoberfläche verglichen. Dass der Adsorptionsprozess auch das Substrat selbst beeinflussen kann, wird durch die Beobachtung einer Phasentrennung bei Proteinadsorption des Lipidgemischs aufgezeigt, das negativ geladene Lipide enthält. / Although it is commonly accepted that the exposition of proteins to man-made materials typically results in protein adsorption on the material surface, little is known about the interplay between the protein-surface interactions involved and the resulting conformational changes of the adsorbing protein. In this study the site-directed spin labeling (SDSL) approach has been extended to the investigation of proteins adsorbed to planar surfaces. The method involves the selective introduction of an artificial spin-labeled side-chain to a predefined residue of the amino acid sequence and allows the determination of the structure and dynamics of proteins by analysis of the electron paramagnetic resonance (EPR) spectra. The globular protein T4 Lysozyme (T4L) has been adsorbed to planar model surfaces to study the correlation between conformational changes of the protein and the physical and chemical properties of the surfaces. Tethering T4L to a planar quartz-supported zwitterionic lipid bilayer shows only minor changes in the structure of the protein. Furthermore, a macroscopic order of the adsorbed protein layer is proven by angular-dependent EPR spectra which allow the determination of the protein orientation. Offering surfaces that are net negatively charged to the highly positively charged T4L leads to the observation of more drastic conformational changes. Here, the conformation of T4L adsorbing to a fluid quartz-supported lipid bilayer containing negatively charged lipids is compared to the structure of T4L adsorbed to the negatively charged but rigid quartz surface. The adsorption process may also influence the substrate itself. This can be shown by the phase separation of the negatively charged lipid bilayer upon protein adsorption.