Signal transduction is critical during the lifetime of a neuron as it navigates to reach its targets, forms functional synaptic connections and adjusts the molecular architecture of these connections in an activity-dependent manner. Understanding the molecular organisation of components required for neuronal signalling will provide novel biological insight and can contribute to the design of therapeutics for neurodevelopmental and neurodegenerative disorders. The focus of the thesis is on determining mechanistic molecular details of a number of distinct cell surface systems implicated in neuronal signalling. Crystallographic studies on the cell surface complex between Eph receptor A4 and ephrinA5 contributed to understanding how the modes of higher order arrangements of receptors involved in guidance affect signal transduction across the membrane. A set of structural and biophysical studies addressed the proteoglycan regulation of RPTPσ-TrkCtrans-synaptic interaction and contributed to deciphering the principles of the switch from axonal growth to synapse establishment and formation. A crystallographic and biochemical analysis of the neuronal C1q-like family, enabled mapping their interactions with potential synaptic partners, and guided functional studies aimed at elucidating their roles in the maintenance of synaptic integrity. Preliminary work on the neuronal Sigma-1 receptor chaperone laid the foundations for the structural determination of this receptor.

<p>Isotropic bicelles are disc-shaped aggregates of lipids and detergents, and are suitable model systems for high-resolution NMR studies of membrane-interacting peptides. In this thesis the structures for the two peptides motilin and transportan were determined by homonuclear ¹H methods in the presence of bicelles, and the structure of the bovine prion protein peptide (bPrPp) was solved in the presence of DHPC micelles. All of these peptides were found to be largely a-helical when bound to the model membranes. In subsequent experiments both motilin and transportan were shown to reside on the surface of the bicelles, whereas bPrPp is more likely to have a transmembrane configuration. </p><p>NMR translational diffusion experiments revealed that the isotropic bicelles studied here are very large objects compared to what is regularly indicated by high-resolution NMR spectroscopy. Furthermore, these studies showed that all three peptides examined interact strongly with bicelles. Investigation of the NMR-relaxation of labeled sites in the peptides motilin and penetratin demonstrated that the overall rotational correlation times for these peptides do not reflect the bicellar size. Such decoupling of NMR relaxation from the dependence of overall size is also seen for the dynamics of the lipid molecules in the bicelles. It is therefore concluded that the overall size is not the sole determinant of the linewidths in NMR spectra, but that extensive motions within the bicelles also exert significant effects. </p><p>Another interesting observation is that the membrane-bound structures of the peptides motilin, transportan, penetratin and bPrPp are very similar, even though these peptides have very different biological functions. In contrast, considerably more variation is observed in the membrane-positioning and molecular dynamics of these peptides. Since the bicelles have been found to induce differences in membrane positioning and molecular dynamics compared to micelles, these model membranes are likely to be important in order to enhance our understanding of the biological function of membrane interacting peptides.</p>

<p>In natural oxygen-producing photosynthesis solar energy is stored as chemical energy, in carbohydrates, fats and amino acids, using water as electron source. The large transmembrane protein complex, PSII, is the key enzyme in the light-driven reactions. Water oxidation is accomplished by a triad in PSII in which the Mn-cluster plays an important role. In the artificial photosynthetic system, nature’s photosynthesis will be mimicked such that hydrogen, a sustainable energy source, can be produced from solar energy and water alone. Since water oxidiation requires the catalytic activity of a Mn-cluster in photosynthesis, different artificially constructed manganese complexes are investigated. </p><p>The dinuclear ([Mn₂(II,III)L(µ-OAc)₂]ClO₄), where L is the X-anion of 2-(<i>N,N</i>-Bis(2-methylpyridyl)aminomethyl)-6-(<i>N</i>-(3,5-ditert-butylbenzyl-2-hydroxy)-<i>N</i>-(pyridylmethyl)aminomethyl)-4-methylphenol, an unsymmetric ligand with two coordinating phenolate groups, has been studied. The two Mn-ions are linked via a mono-µ-oxo bridge and two acetate ligands. Q-band Electron Paramagnetic Resonance was conducted on the Unsymmetric Mn₂(II,III) Complex. Aquired results show that the complex has a 2600 Gauss broad signal (11 400-14 000 Gauss) with 14-17 lines at g~2 and hyperfines of 120 Gauss. This is consistent with previous X-band studies. Q-band spectra of the Unsymmetric Mn(II,III) display increased hyperfine resolution compared to Qband spectra of the symmetric complex, Mn₂(bpmp)(µ-OAC)₂. This is noticeable since Unsymmetric Mn2(II,III) and Mn₂ (bpmp)(µ-OAC)₂ partly overlap in low-frequency experiments (X-band EPR). </p><p>Further investigations are yet to be expected. Nevertheless, the conducted thesis study provides important knowledge in the futuristic goal of building an artificial super-complex.</p>

Isotropic bicelles are disc-shaped aggregates of lipids and detergents, and are suitable model systems for high-resolution NMR studies of membrane-interacting peptides. In this thesis the structures for the two peptides motilin and transportan were determined by homonuclear 1H methods in the presence of bicelles, and the structure of the bovine prion protein peptide (bPrPp) was solved in the presence of DHPC micelles. All of these peptides were found to be largely a-helical when bound to the model membranes. In subsequent experiments both motilin and transportan were shown to reside on the surface of the bicelles, whereas bPrPp is more likely to have a transmembrane configuration. NMR translational diffusion experiments revealed that the isotropic bicelles studied here are very large objects compared to what is regularly indicated by high-resolution NMR spectroscopy. Furthermore, these studies showed that all three peptides examined interact strongly with bicelles. Investigation of the NMR-relaxation of labeled sites in the peptides motilin and penetratin demonstrated that the overall rotational correlation times for these peptides do not reflect the bicellar size. Such decoupling of NMR relaxation from the dependence of overall size is also seen for the dynamics of the lipid molecules in the bicelles. It is therefore concluded that the overall size is not the sole determinant of the linewidths in NMR spectra, but that extensive motions within the bicelles also exert significant effects. Another interesting observation is that the membrane-bound structures of the peptides motilin, transportan, penetratin and bPrPp are very similar, even though these peptides have very different biological functions. In contrast, considerably more variation is observed in the membrane-positioning and molecular dynamics of these peptides. Since the bicelles have been found to induce differences in membrane positioning and molecular dynamics compared to micelles, these model membranes are likely to be important in order to enhance our understanding of the biological function of membrane interacting peptides.

In natural oxygen-producing photosynthesis solar energy is stored as chemical energy, in carbohydrates, fats and amino acids, using water as electron source. The large transmembrane protein complex, PSII, is the key enzyme in the light-driven reactions. Water oxidation is accomplished by a triad in PSII in which the Mn-cluster plays an important role. In the artificial photosynthetic system, nature’s photosynthesis will be mimicked such that hydrogen, a sustainable energy source, can be produced from solar energy and water alone. Since water oxidiation requires the catalytic activity of a Mn-cluster in photosynthesis, different artificially constructed manganese complexes are investigated. The dinuclear ([Mn2(II,III)L(µ-OAc)2]ClO4), where L is the X-anion of 2-(N,N-Bis(2-methylpyridyl)aminomethyl)-6-(N-(3,5-ditert-butylbenzyl-2-hydroxy)-N-(pyridylmethyl)aminomethyl)-4-methylphenol, an unsymmetric ligand with two coordinating phenolate groups, has been studied. The two Mn-ions are linked via a mono-µ-oxo bridge and two acetate ligands. Q-band Electron Paramagnetic Resonance was conducted on the Unsymmetric Mn2(II,III) Complex. Aquired results show that the complex has a 2600 Gauss broad signal (11 400-14 000 Gauss) with 14-17 lines at g~2 and hyperfines of 120 Gauss. This is consistent with previous X-band studies. Q-band spectra of the Unsymmetric Mn(II,III) display increased hyperfine resolution compared to Qband spectra of the symmetric complex, Mn2(bpmp)(µ-OAC)2. This is noticeable since Unsymmetric Mn2(II,III) and Mn2 (bpmp)(µ-OAC)2 partly overlap in low-frequency experiments (X-band EPR). Further investigations are yet to be expected. Nevertheless, the conducted thesis study provides important knowledge in the futuristic goal of building an artificial super-complex.

No description available.

Biological membranes are quasi two-dimensional self-assembled structure, primarily serving as a barrier to the leakage of cell’s contents. The main constituents of biological membrane are various amphiphilic lipids that form bilayers in an aqueous environment. These lipids carry acidic and/or basic functional groups that ionize in water, giving some of them a net electrical charge. Such a lipid molecule, when integrated into the membrane, experiences electrostatic forces from all other charged objects around it, including ions, surrounding lipids, and other molecules such as cationic peptides. The electrostatic interaction can profoundly influence the membrane, to which many phenomena with physiological significance as well as biophysical interest can be ascribed. In this thesis, we concentrate on investigating the electrostatic properties of lipid membranes. First, we study how the electrostatic interaction affects their preferred structure. To this end, we adopt a coarse-grain model that preserves the dominant characteristics of the lipids, in which the electrostatic interaction is treated within the “renormalized” Debye-H¨uckel theory. In particular, we calculate the spontaneous curvature of a phospholipid monolayer, along with other associated quantities. Our results suggest that such divalent ions as Mg2+ can stabilize HII phases of lipids (inverted hexagonal phases), which would otherwise form lamellar phases. Second,we investigate the competitive binding of ions and cationic peptides onto a monolayer of lipopolysaccharide (LPS) molecules, a class of highly charged bio-molecules found in the outer leaflet of the outer membranes of gram-negative (G-) bacteria. Cationic anti-microbial peptides (AMPs) can selectively kill bacteria, and it is suggested that they destabilize the LPS layer, easing their permeation across it, a process of great physiological and clinical interest. To this end, we model the LPS layer as a collection of charged “binding sites”, based on which we study the binding of cations (monovalent and divalent) and cationic peptides onto the layer. Our calculations suggest that the peptides can compete with divalent ions on the binding to the layer. It has been empirically known that since the stability of an LPS layer relies greatly on the bridging of divalent ions, the substitution of these ions by the peptides significantly compromises its stability. Our results offer a quantitative basis for this observation, thus providing a possible mechanism of an important step in the action of AMPs against G- bacteria.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Computational Biology and Molecular Biophysics." Includes bibliographical references (p. 210-226).

Biological membranes are quasi two-dimensional self-assembled structure, primarily serving as a barrier to the leakage of cell’s contents. The main constituents of biological membrane are various amphiphilic lipids that form bilayers in an aqueous environment. These lipids carry acidic and/or basic functional groups that ionize in water, giving some of them a net electrical charge. Such a lipid molecule, when integrated into the membrane, experiences electrostatic forces from all other charged objects around it, including ions, surrounding lipids, and other molecules such as cationic peptides. The electrostatic interaction can profoundly influence the membrane, to which many phenomena with physiological significance as well as biophysical interest can be ascribed. In this thesis, we concentrate on investigating the electrostatic properties of lipid membranes. First, we study how the electrostatic interaction affects their preferred structure. To this end, we adopt a coarse-grain model that preserves the dominant characteristics of the lipids, in which the electrostatic interaction is treated within the “renormalized” Debye-H¨uckel theory. In particular, we calculate the spontaneous curvature of a phospholipid monolayer, along with other associated quantities. Our results suggest that such divalent ions as Mg2+ can stabilize HII phases of lipids (inverted hexagonal phases), which would otherwise form lamellar phases. Second,we investigate the competitive binding of ions and cationic peptides onto a monolayer of lipopolysaccharide (LPS) molecules, a class of highly charged bio-molecules found in the outer leaflet of the outer membranes of gram-negative (G-) bacteria. Cationic anti-microbial peptides (AMPs) can selectively kill bacteria, and it is suggested that they destabilize the LPS layer, easing their permeation across it, a process of great physiological and clinical interest. To this end, we model the LPS layer as a collection of charged “binding sites”, based on which we study the binding of cations (monovalent and divalent) and cationic peptides onto the layer. Our calculations suggest that the peptides can compete with divalent ions on the binding to the layer. It has been empirically known that since the stability of an LPS layer relies greatly on the bridging of divalent ions, the substitution of these ions by the peptides significantly compromises its stability. Our results offer a quantitative basis for this observation, thus providing a possible mechanism of an important step in the action of AMPs against G- bacteria.

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Computational Biology and Molecular Biophysics." Includes bibliographical references.