Zugl.: Dresden, Techn. Univ., Diss., 2005

Universiẗat-Gesamthochsch., Diss.--Paderborn, 1996.

Techn. Univ., Diss., 2005--Darmstadt

The interactions between cholesterol and other membrane molecules determine important membrane properties. It was shown that even small changes in the molecular structure of cholesterol have a crucial influence on these interactions. We recently reported that in addition to alterations in the tetracyclic ring structure, the iso-branched side chain of cholesterol also has a significant impact on membrane properties (Scheidt H. et al. 2013 Angew. Chem. Int. Ed. Engl. 52, 12848-12851). Here we used synthetic cholesterol analogs to investigate the influence of an unbranched aliphatic side chain of different length. The 2H NMR order parameter of the phospholipid chains and therefore the molecular packing of the phospholipid molecules shows a significant dependence on the sterol’s alkyl side chain length, while , membrane permeation studied by a dithionite ion permeation assay and lateral diffusion measured by 1H MAS pulsed field gradient NMR are less influenced. To achieve the same molecular packing effect similar to that of an iso-branched aliphatic side chain, a longer unbranched side chain (n-dodecyl instead of n-octyl) at C17 of cholesterol is required. Obviously, sterols having a branched iso- alkyl chain with two terminal methyl groups exhibit altered cholesterol-phospholipid-interactions compared to analogous molecules with a straight unbranched chain.

In contrast to the static snapshots provided by protein crystallography, G protein-coupled receptors constitute a group of proteins with highly dynamic properties, which are required in the receptors’ function as signaling molecule. Here, the human neuropeptide Y2 receptor was reconstituted into a model membrane composed of monounsaturated phospholipids and solid-state NMR was used to characterize its dynamics. Qualitative static 15N NMR spectra and quantitative determination of 1H-13C order parameters through measurement of the 1H-13C dipolar couplings of the CH, CH2 and CH3 groups revealed axially symmetric motions of the whole molecule in the membrane and molecular fluctuations of varying amplitude from all molecular segments. The molecular order parameters (Sbackbone = 0.59-0.67, SCH2 = 0.41-0.51 and SCH3 = 0.22) obtained in directly polarized 13C NMR experiments demonstrate that the Y2 receptor is highly mobile in the native-like membrane. Interestingly, according to these results the receptor was found to be slightly more rigid in the membranes formed by the monounsaturated phospholipids than by saturated phospholipids as investigated previously. This could be caused by an increased chain length of the monounsaturated lipids, which may result in a higher helical content of the receptor. Furthermore, the incorporation of cholesterol, phosphatidylethanolamine, or negatively charged phosphatidylserine into the membrane did not have a significant influence on the molecular mobility of the Y2 receptor.

The interactions between cholesterol and other membrane molecules determine important membrane properties. It was shown that even small changes in the molecular structure of cholesterol have a crucial influence on these interactions. We recently reported that in addition to alterations in the tetracyclic ring structure, the iso-branched side chain of cholesterol also has a significant impact on membrane properties (Scheidt H. et al. 2013 Angew. Chem. Int. Ed. Engl. 52, 12848-12851). Here we used synthetic cholesterol analogs to investigate the influence of an unbranched aliphatic side chain of different length. The 2H NMR order parameter of the phospholipid chains and therefore the molecular packing of the phospholipid molecules shows a significant dependence on the sterol’s alkyl side chain length, while , membrane permeation studied by a dithionite ion permeation assay and lateral diffusion measured by 1H MAS pulsed field gradient NMR are less influenced. To achieve the same molecular packing effect similar to that of an iso-branched aliphatic side chain, a longer unbranched side chain (n-dodecyl instead of n-octyl) at C17 of cholesterol is required. Obviously, sterols having a branched iso- alkyl chain with two terminal methyl groups exhibit altered cholesterol-phospholipid-interactions compared to analogous molecules with a straight unbranched chain.

In contrast to the static snapshots provided by protein crystallography, G protein-coupled receptors constitute a group of proteins with highly dynamic properties, which are required in the receptors’ function as signaling molecule. Here, the human neuropeptide Y2 receptor was reconstituted into a model membrane composed of monounsaturated phospholipids and solid-state NMR was used to characterize its dynamics. Qualitative static 15N NMR spectra and quantitative determination of 1H-13C order parameters through measurement of the 1H-13C dipolar couplings of the CH, CH2 and CH3 groups revealed axially symmetric motions of the whole molecule in the membrane and molecular fluctuations of varying amplitude from all molecular segments. The molecular order parameters (Sbackbone = 0.59-0.67, SCH2 = 0.41-0.51 and SCH3 = 0.22) obtained in directly polarized 13C NMR experiments demonstrate that the Y2 receptor is highly mobile in the native-like membrane. Interestingly, according to these results the receptor was found to be slightly more rigid in the membranes formed by the monounsaturated phospholipids than by saturated phospholipids as investigated previously. This could be caused by an increased chain length of the monounsaturated lipids, which may result in a higher helical content of the receptor. Furthermore, the incorporation of cholesterol, phosphatidylethanolamine, or negatively charged phosphatidylserine into the membrane did not have a significant influence on the molecular mobility of the Y2 receptor.

Das Verstaendnis dafuer, welche Freiheitsgrade fuer das Eingeschlossensein der Quarks von Bedeutung sind, ist ein altbekanntes Problem. Da weithin angenommen wird, dass das Zentrum der Eichgruppe eine bedeutende Rolle spielt, ist es interessant, eine Theorie mit einem trivialen Zentrum zu untersuchen. Das einfachste Modell, um dieses Problem zu untersuchen, ist eine Theorie mit ungeradzahliger Dimension der Darstellung der Eichgruppe SU(2). Theorien mit einem trivialen Zentrum werden schon seit langer Zeit in zwei verschiedenen Diskretisierungen untersucht: die adjungierte Wilson-Wirkung und die Villain-Wirkung. Es stellte sich heraus, dass sie aus zweierlei Gruenden problematisch sind. Zunaechst zeigte sich, dass in beiden Fällen ein bulk-Phasenuebergang den physikalischen Phasenuebergang bei endlicher Temperatur ueberschattet. Darueberhinaus erwies es sich im Falle der Villain-Theorie, dass die Anwesenheit von Twist-Sektoren fuer die Konstruktion eines ergodischen Algorithmus problematisch sein kann. Die Gitter-Artefakte, die den bulk-Phasenuebergang verursachen, wurden mit Z(2) Monopolen identifiziert. Diese Monopole koennen mit Hilfe eines entsprechenden chemischen Potentials unterdrueckt werden. Eine erste Untersuchung des Phasenuebergangs bei endlicher Temperatur durch andere Autoren wurde nur im Falle der Villain-Wirkung durchgefuehrt, wobei in dieser Untersuchung die Twist-Sektoren ohne Beruecksichtigung blieben. In der vorliegenden Arbeit untersuchen wir nichtstoerungstheoretisch die Wilson-Wirkung in der adjungierten Darstellung der Eichgruppe SU(2) mit einem chemischen Potential, welches die Z(2)-Monopole bei nicht verschwindender Temperatur und bei Temperatur Null unterdrueckt. Wir untersuchen hierbei die Auswirkungen des chemischen Potentials lambda auf einige Observable. Fuer hinreichend grosse lambda zeigen die Observablen keine Diskontinuitaet in der adjungierten Kopplung. In diesem Gebiet des Phasendiagramms untersuchen wir, meist eingeschraenkt auf den trivialen Twist-Sektor, die Existenz eines Phasenuebergangs bei endlicher Temperatur. Um diesen Phasenuebergang zu identifizieren, gelingt es uns, einen neuen Ordungsparameter zu definieren, den wir erfolgreich auch in der fundamentalen Darstellung der SU(2) testen. Ferner analysieren wir die raeumliche Verteilung der fundamentalen Polyakov-loop-Variable und des Pisaer Unordnungs-Operators, welcher die Kondensation magnetischer Ladungen beschreibt. Die Ergebnisse, die wir mit diesen Untersuchungsmethoden erhielten, lassen auf einen vom bulk-Phasenuebergang entkoppelten Phasenuebergang bei endlicher Temperatur oder einen cross-over schliessen. / The understanding of which degrees of freedom are relevant for the confinement of quarks is a long standing problem. Since it is widely believed that the center of the gauge group plays an important role, it is interesting to study a theory with a trivial center. The simplest model to investigate this problem is provided by a theory in an odd-dimensional representation of the gauge group SU(2). Center-blind theories were studied long time ago in two different discretizations, the adjoint Wilson and the Villain action, and they turned out to be problematic for two reasons. In both cases a bulk phase transition was shown to overshadow the physical finite temperature one. Another feature, pointed out in the Villain case, was the presence of twist sectors, which could cause difficulties in the construction of an ergodic algorithm. The lattice artifacts responsible for the bulk phase transition were identified with Z(2) monopoles and they could be suppressed by the use of an appropriate chemical potential. A preliminary investigation of the finite temperature phase transition by other authors was done only in the Villain case and without taking care of the twist sectors. In this thesis we perform a lattice study of the Wilson action in the adjoint representation of the gauge group SU(2) with a chemical potential, which suppresses the Z(2) monopoles at zero and non-zero temperature. We investigate the effects of the chemical potential lambda on some observables. For large enough lambda at vanishing temperature the observables do not show any discontinuity in the adjoint coupling. In this region we study the existence of a finite temperature phase transition restricting ourselves mainly to the trivial twist sector. In order to detect this phase transition we are able to define a new order parameter, which we successfully test also for the case of the fundamental representation of SU(2). Furthermore we analyze the spatial distribution of the fundamental Polyakov loop and the Pisa disorder operator which detects the condensation of magnetic charges. These different tools provide an indication for a finite temperature phase transition or crossover decoupled from the bulk phase transition.

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance – PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol – using 2H solidstate nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance – PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol – using 2H solidstate nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.