Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 04/06 Archiv

The focus of this thesis concerns the morphology control of ordered mesoporous carbon (OMC) materials. Ordered mesoporous carbons with diverse morphologies, that are thin films, fibers – embedded in anodic alumina membranes and free-standing – or spherical nanoparticles, have been successfully prepared by soft-templating procedures. The mechanisms of structure formation and processing were investigated with in-situ SAXS measurements and their application in high capacity lithium-sulfur batteries was successfully tested in cooperation with Guang He and Linda Nazar from the University of Waterloo in Canada. The Li-S batteries receive increasing attention due to their high theoretical energy density which is 3 to 5 times higher than from lithium-ion batteries. For this type of battery the specific pore volume is crucial for the content of the active component (sulfur) in the cathode and therefore correlates with the capacity and gravimetric energy density of the battery. At first, mesoporous thin films with 2D-hexagonal structure were obtained through organic-organic self-assembly of a preformed oligomeric resol precursor and the triblock copolymer template Pluronic P123. The formation of a condensed-wall material through thermopolymerization of the precursor oligomers resulted in mesostructured phenolic resin films. Subsequent decomposition of the surfactant and partial carbonization were achieved through thermal treatment in inert atmosphere. The films were crack-free with tunable homogenous thicknesses, and showed either 2D-hexagonal or lamellar mesostructure. An additional, yet unknown 3D-mesostructure was also found. In the second part, cubic and circular hexagonal mesoporous carbon phases in the confined environment of tubular anodic alumina membrane (AAM) pores were obtained by self-assembly of the mentioned resol precursor and the triblock copolymer templates Pluronic F127 or P123, respectively. Casting and solvent-evaporation were also followed by thermopolymerization, thermal decomposition of the surfactant and carbonization through thermal treatment at temperatures up to 1000 °C in an inert atmosphere. For both structures the AAM pores were completely filled and no shrinkage was observed, due to strong adhesion of the carbon wall material to the AAM pore walls. As a consequence of this restricted shrinkage effect, the mesophase system stayed almost constant even after thermal treatment at 1000 °C, and pore sizes of up to 20 nm were obtained. In the third part, the aforementioned mesoporous films and embedded fibers in AAMs were further investigated concerning structure formation and carbonization in an in-situ SAXS study. The in-situ measurements revealed that for both systems the structure formation occurs during the thermopolymerization step. Therefore the process of structure formation differs significantly from the known evaporation-induced self-assembly (EISA) and may rather be viewed as thermally-induced self-assembly. As a result, the structural evolution strongly depends on the chosen temperature, which controls both the rate of the mesostructure formation and the spatial dimensions of the resulting mesophase. In the fourth part the syntheses recipes for AAMs were applied on a presynthesized silica template for synthesis of freestanding mesoporous carbon nanofibers. The syntheses start with casting of carbon nanofibers with a silica precursor solution leading to a porous silica template after calcination with tubular pores mimicking the initial carbon nanofibers. A synthesis concept using triconstituent coassembly of resol, tetraethylorthosilicate as additional silica precursor and Pluronic F127 was applied here. The silica from the additional precursor was found to be beneficial, due to reduced shrinkage and created additional porosity after etching it. Those OMC nanofibers therefore exhibited a very large surface area and a high pore volume of 2486 m2/g and 2.06 cm3/g, respectively. Due to their extremely high porosity values, those fibers were successfully applied as sulfur host and electrode material in lithium-sulfur batteries. The fifth and last part focuses on the synthesis of spherical mesoporous carbon nanoparticles. Therefore the triconstituent coassembly was applied on a silica template with spherical pores, which was derived from the opal structure of colloidal crystals made from 400 nm PMMA spheres. The spherical ordered mesoporous carbon nanoparticles feature extremely high inner porosity of 2.32 cm3/g and 2445 m2/g, respectively They were successfully applied as cathode material in Li-S batteries, where they showed high reversible capacity up to 1200 mAh/g and good cycle efficiency. The final product consists of spherical mesoporous carbon particles with a diameter of around 300 nm and 2D-hexagonal porosity. The particles could be completely separated by sonification to form stable colloidal suspensions. This could be the base for further applications such drug delivery.

The INO80 complex is a chromatin remodeler involved in diverse nuclear processes like transcriptional regulation, replication fork progression, checkpoint regulation and DNA double strand break repair. In the yeast S. cerevisiae the complex consists of 15 subunits with a total molecular mass of about 1.2 MDa. Knowledge about the atomic structure and molecular architecture of the entire complex is scarce. Similarly, an understanding for the roles of the individual subunits of the complex is mostly lacking. Especially the function of actin and the actin related proteins Arp4 and Arp8 which are found as monomeric components of INO80 and other chromatin remodelers is poorly understood. The goal of this study was to elucidate the functional architecture of the INO80 complex by using a hybrid methods approach. Different structural techniques such as X-ray crystallography, small angle X-ray scattering (SAXS) and electron microscopy (EM) were combined to achieve this goal. Additionally, various functional assays to study the biochemical properties of the actin related proteins and their interaction with actin were employed. In a set of primary experiments expression and purification protocols for seven individual INO80 components, namely Arp4, Arp5, Arp8, Ies4, Ies5, Ies6 and Nhp10 could be established. Additionally, four subcomplexes containing more than one protein, namely Rvb1-Rvb2, Nhp10-Ies5, Nhp10-Ies5-Ies3 and Arp5-Ies6 were purified. Thereby two previously unknown interactions between the INO80 subunits Nhp10 and Ies5, as well as Arp5 and Ies6 could be identified. Subsequently, the newly identified complexes of Nhp10-Ies5-Ies3 and Arp5-Ies6 were studied with SAXS to obtain low resolution solution structures of both. On top of that the entire INO80 complex was purified endogenously from S. cerevisiae and studied by EM. Unfortunately, a three dimensional reconstruction of the remodeler could not be created. Crystallization attempts on all purified INO80 components were successful for the complex of Rvb1-Rvb2 and the actin related protein Arp4. Whereas the structure of Rvb1-Rvb2 could not be solved due to limited diffraction an atomic structure of ATP bound Arp4 at 3.4 Å resolution was obtained. Remarkably, Arp4 does not form filaments despite its high similarity to conventional actin. The lack of polymerization is confirmed by the SAXS structure of isolated Arp4 which indicates it to be monomeric and can be nicely explained on the basis of the crystal structure. Several loop insertions and deletions at positions which are crucial for contact formation within the actin filament, especially at the pointed end of the molecule, prevent Arp4 to engage in filament like interactions. Furthermore, the crystal structure of Arp4 reveals an ATP molecule to be constitutively bound to the protein. The lack of ATPase activity of Arp4 in contrast to actin can be explained with the help of the crystal structure as well. Several residues in the nucleotide clamping loops of Arp4 are divergent from actin leading to a tighter closure and better shielding of the phosphate moieties of the bound ATP from the environment. Most interestingly, Arp4 dramatically influences actin polymerization kinetics. Different fluorescence assays and in vitro TIRF microscopy were used to show that Arp4 is able to inhibit actin polymerization and to depolymerize actin filaments most likely by complex formation with monomeric ADP-actin via the barbed end. Its ability to inhibit actin filament nucleation without sequestering actin while still allowing ADP to ATP exchange within actin resembles the actin binding protein profilin. Arp8 was confirmed by SAXS measurements to be monomeric as well. It is able to sequester actin monomers and to slowly depolymerize actin filaments. Consistent with the formation of a discrete Arp4-Arp8-actin complex within the INO80 remodeler the effects of Arp4 on actin polymerization are further stimulated by Arp8. As both proteins reciprocally enhance their individual effects on actin it is likely that they help to maintain actin in a defined monomeric state within the INO80 chromatin remodeler. The data further suggest a possible assembly between actin and Arp4 via their barbed ends and a model how the Arp4-Arp8-actin complex is integrated into the INO80 chromatin remodeler. Taken together, the findings represent a remarkable advancement in the understanding of nuclear actin related proteins and nuclear actin biochemistry in general. Most excitingly, they indicate a link between chromatin remodeling and nuclear actin dynamics possibly giving chromatin remodeling complexes a role in the actin mediated large scale movement of chromatin.

Recombinant proteins gain more and more importance in the pharmaceutical market. One major challenge is to assure the protein integrity during manufacturing, shipping and storage. Complementary analytical approaches are deemed necessary to fully characterize and understand structural changes of the protein and the quality of formed aggregates. Hence, the focus of this thesis was the investigation of protein unfolding and protein-protein interactions as first steps of protein aggregation. Using extrinsic fluorescence analysis and B22 techniques, protein unfolding as well as attractive or repulsive protein-protein interactions could be determined for both the protein aggregates as well as the monomer, due to prior size-separation, simultaneously and individually. As both fluorescence and B22 analyses can be conducted in combination with SEC, the most widely used and best established technique, the developed methods represent easily accessible and complementary approaches to analyze protein unfolding, interactions and aggregation. In sum, this allows to shed more light on the induction factors and the sequence of events in protein aggregation during formulation development, manufacturing and storage.

Ribosomes are macromolecular protein-RNA complexes translating mRNA into protein. To date, crystal structures are available for the bacterial 30S and archaeal 50S subunits, as well as the complete bacterial 70S ribosomes. Eukaryotic ribosomes are much more complex in terms of ribosomal RNA and proteins. However, to date high-resolution crystal structures of eukaryotic ribosomes or ribosomal subunits are lacking. In order to build reliable models for the eukaryotic rRNA, we developed an approach for large scale homology and de novo modeling of RNA and subsequent exible tting into high-resolution cryo-EM density maps. Using this approach we built a model of the T. aestivum and the S. cerevisiae ribosome based on available cryo-EM maps at 5.5 Å and 6.1 Å resolution, respectively. The model comprises of 98% of the eukaryotic rRNA including all 21 RNA expansion segments (ES) and structurally six variable regions. Further, we were able to localize 74/80 (92.5%) of the ribosomal proteins. The model reveals unique ES-ES and r-protein-ES interactions, providing new insight into the structure and evolution of the eukaryotic ribosome. Moreover, the model was used for analyzing functional ribosomal complexes, i.e. the characterization of dierent nascent polypeptide chains within the ribosomal tunnel, intermediates of protein translocation as well as mRNA quality control.