Podcasts about saxs

Hello and welcome to another episode of Leaders in Tech, where we shine a spotlight on the trailblazers revolutionizing the world of technology. Today, we have the privilege of speaking with Scott Bendle, the Chief Information Officer (CIO) of Raku, a company specializing in x-ray analytical equipment for industries ranging from pharmaceuticals to semiconductors. Scott shares his unique journey from quality engineer to CIO, offering insights into leadership, adaptability, and the future of tech. Let's dive into Scott's incredible story and the lessons we can learn from it.Here's more about Scott Bendle:●Highly motivated and experienced business leader with expert proficiency in defining and guiding information systems vision, strategy, and execution for SMB organizations.●An effective subject matter expert, capable of transforming business needs into technological solutions, adding value through the focused application of suitable technology while engaging in large-scale IT operations management.●Regarded for the ability to deliver outstanding results with the highest degree of expertise and professionalism.Areas of Expertise:●Technology Leadership● Project Management● Team Leadership● Process Improvement●IT Strategy and Vision● Operations Management● Critical Thinking● IS/QS 9000 ImplementationCareer Achievements:●Rigaku Americas Corporation. Oversees network services used by 450 employees across the United States, United Kingdom, Germany, and Poland. Successfully managed through a crisis scenario where all network knowledge and passwords were lost, assembling a team of contractors to regain access and restructure the network as a scalable resource.●Rigaku Innovative Technologies. Managed operations, with authority over departments that included Engineering, Manufacturing, Deposition, Facilities, Production Control, and Shipping and Receiving.●Magnequench International Inc. Excelled in labor relations, managing a unionized workforce in a 24/7 operating environments; managed 225 production team members in a $125MM manufacturing company.Company description: Since its inception in 1951, Rigaku has been at the forefront of analytical and industrial instrumentation technology. Today, with hundreds of major innovations to their credit, the Rigaku Group of Companies are world leaders in the fields of general X-ray diffraction (XRD), thin film analysis (XRF, XRD and XRR), X-ray fluorescence spectrometry (TXRF, EDXRF and WDXRF), small angle X-ray scattering (SAXS), protein and small molecule X-ray crystallography, Raman spectroscopy, X-ray optics, semiconductor metrology (TXRF, XRF, XRD and XRR), X-ray sources, computed tomography, nondestructive testing and thermal analysis.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.18.533291v1?rss=1 Authors: Ptak, C. P., Peterson, T. A., Hopkins, J. B., Ahern, C. A., Shy, M. E., Piper, R. C. Abstract: Mutations in Myelin Protein Zero (MPZ) account for 5% of Charcot-Marie-Tooth cases and can cause demyelinating or axonal phenotypes, reflecting the diverse roles of MPZ in Schwann cells. MPZ holds the apposing membranes of the myelin sheath together, with the adhesion role fulfilled by the extracellular Immunoglobulin-like domain (IgMPZ), which can oligomerize. Current knowledge for how the IgMPZ might form oligomeric assemblies involving 3 weakly-interacting interfaces has been extrapolated from a protein crystal structure in which individual rat IgMPZ subunits are packed together under artificial conditions. These interfaces include one that organizes the IgMPZ into tetramers, a 'dimer' interface that could link tetramers together, and a third hydrophobic interface that could mediate binding to lipid bilayers or the same hydrophobic surface on another IgMPZ domain. There are at present no data confirming whether the proposed IgMPZ interfaces actually mediate oligomerization in solution, whether they are required for the adhesion activity of MPZ, whether they are important for myelination, or whether their loss results in disease. We performed NMR and SAXS analysis of wild-type IgMPZ as well as mutant forms with amino-acid substitutions designed to interrupt its presumptive oligomerization interfaces. Here, we confirm the interface that mediates IgMPZ tetramerization, but find that dimerization is mediated by a distinct interface that has yet to be identified. We next correlated CMT phenotypes to subregions within IgMPZ tetramers. Axonal late-onset disease phenotypes (CMT2I/J) map to surface residues of IgMPZ proximal to the transmembrane domain. Early-onset demyelinating disease phenotypes (CMT1B/Dejerine-Sottas syndrome) map to two groups: one is described by variants that disrupt the stability of the Ig-fold itself and are largely located within the core of the Ig domain; whereas another describes a surface on the distal outer surface of IgMPZ tetramers. Computational docking studies predict that this latter disease-relevant subregion may mediate dimerization of IgMPZ tetramers. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.04.509781v1?rss=1 Authors: Menzel, M., Grässel, D., Rajkovic, I., Zeineh, M. M., Georgiadis, M. Abstract: Disentangling human brain connectivity requires an accurate description of neuronal trajectories. However, a detailed mapping of axonal orientations is challenging because axons can cross one another on a micrometer scale. Diffusion magnetic resonance imaging (dMRI) can be used to infer neuronal connectivity because it is sensitive to axonal alignment, but it has limited resolution and specificity. Scattered Light Imaging (SLI) and small-angle X-ray scattering (SAXS) reveal neuronal orientations with microscopic resolution and high specificity, respectively. Here, we combine both techniques to achieve a cross-validated framework for imaging neuronal orientations, with comparison to dMRI. We evaluate brain regions that include unidirectional and crossing fiber tracts in human and vervet monkey brains. We find that SLI, SAXS, and dMRI all agree regarding major fiber pathways. SLI and SAXS further quantitatively agree regarding fiber crossings, while dMRI overestimates the amount of crossing fibers. In SLI, we find a reduction of peak distance with increasing out-of-plane fiber angles, confirming theoretical predictions, validated against both SAXS and dMRI. The combination of scattered light and X-ray imaging can provide quantitative micrometer 3D fiber orientations with high resolution and specificity, enabling detailed investigations of complex tract architecture in the animal and human brain. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.30.510198v1?rss=1 Authors: Georgiadis, M., Menzel, M., Reuter, J. A., Born, D., Kovacevich, S., Alvarez, D., Gao, Z., Guizar-Sicairos, M., Weiss, T. M., Axer, M., Rajkovic, I., Zeineh, M. M. Abstract: Myelinated axons (nerve fibers) efficiently transmit signals throughout the brain via action potentials. Multiple methods that are sensitive to axon orientations, from microscopy to magnetic resonance imaging, aim to reconstruct the brain's structural connectome. As billions of nerve fibers traverse the brain with various possible geometries at each point, resolving fiber crossings is necessary to generate accurate structural connectivity maps. However, doing so with specificity is a challenging task because signals stemming from oriented fibers can be influenced by brain (micro)structures unrelated to myelinated axons. X-ray scattering can specifically probe myelinated axons due to the periodicity of the myelin sheath, which yields distinct peaks in the scattering pattern. Here, we show that scanning small-angle X-ray scattering (SAXS) can be used to detect myelinated, axon-specific fiber crossings. We first demonstrate the capability using strips of human corpus callosum to create artificial double and triple crossing fiber geometries, and then apply the method in mouse, pig, vervet monkey, and human brains. Given its specificity, capability of 3-dimensional sampling and high resolution, scanning SAXS can serve as a ground truth for validating MRI as well as microscopy-based methods. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

In this episode Pranoti sits down with Brian R. Pauw, who is a researcher at BAM Federal Institute for Materials Research and Testing, Germany. They take a deeper dive into Brian's career journey spanning four countries. He shares his passion for finding the perfect X-ray scattering data (correction) and more equity in science. Find out more at thesciencetalk.com/real-scientists-nano/curators/

This week's real nano scientist is Brian R. Pauw. He will take us on a journey to the fine structure of materials at the nanoscale. Follow along @RealSci_Nano for tweets about X-ray scattering, a virtual tour to the lab (and Berlin!) and more! Find out more at thesciencetalk.com/real-scientists-nano/curators/

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.14.363911v1?rss=1 Authors: Li, J., Huang, Q. Abstract: Oat protein isolate (OPI) is among the plant proteins with valuable functionalities (e.g. emulsification) during daily supplement intake. Understanding their structures helps to manipulate oat proteins at small scale, which enables the appropriate deployment of their functions. Based upon such understanding, the molecular structure of oat protein isolate (OPI) in aqueous medium was investigated by synchrotron small angle X ray scattering (SAXS), and this study allows a structural reconstitution of OPI in aqueous medium. Besides, this SAXS study is complimentary to the previous study (Liu et al. J. Agric. Food Chem. 2009, 57, 4552 to 4558). From form factor fitting, we confirmed that OPI aqueous solutions at low concentrations (0.3~2 mg/mL) obtained a disk conformation (41.4*41.4*10.2 A3). Once protein concentration increased to 5 mg/mL and 10 mg/mL, the individual disk proteins formed large dimensional rod like aggregates, which was evidenced by the analyses of effective structure factor and pair distribution function (PDF). Based on the PDF results, the ab initio models of OPI particles at low concentrations (0.3 mg/mL to 2.0 mg/mL) were restored by using GASBOR algorithm. Finally, we found that weak attraction between OPI particles occurred, which was verified by second virial coefficient and pair potential. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.03.367284v1?rss=1 Authors: Brittain, T. J., O'Malley, M. C., Swaim, C. M., Fink, R. A., Kokhan, O. Abstract: C-type cytochromes play an important role in respiration of dissimilatory metal-reducing bacteria. They form extended conduits for charge transfer between the cellular metabolism and external electron acceptors such as particles of iron oxide, metal ions, and humic substances. Out of more than a hundred c-type cytochromes in Geobacter sulfurreducens, only a small fraction has been previously characterized. Here we present our results on expression and biophysical characterization of GSU0105, a novel 3-heme cytochrome, important for Fe(III) respiration in G. sulfurreducens. We successfully cloned the gene and achieved ~3 mg/L of culture GSU0105 expression in E.coli. Despite a similar size (71 amino acids) and the same number of c-type hemes to the members of the cytochrome (cyt) c7 family, multiple sequence alignment suggests that GSU0105 does not belong to the cyt c7 family. UV-Vis spectroscopy revealed typical c-type cytochrome spectral features, including a weak iron-sulfur charge transfer band suggesting that at least one heme is ligated with a methionine residue. Far UV circular dichroism studies demonstrate approximately 35% content of -helices and {beta}-sheets, each, as well as thermal aggregation occurring above 60C. A combination of SAXS and analytical size exclusion chromatography data shows that GSU0105 is monomeric in solution. Finally, affinity pull-down assays demonstrate high binding affinity to PpcD and weaker binding to the other members of the cyt c7 family. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.28.359091v1?rss=1 Authors: Senicourt, L., le Maire, A., Allemand, F., Carvalho, J. E., Guee, L., Germain, P., Schubert, M., Bernado, P., Bourguet, W., Sibille, N. Abstract: Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) form heterodimers that activate target gene transcription by recruiting co-activator complexes in response to ligand binding. The nuclear receptor (NR) co-activator TIF2 mediates this recruitment by interacting with the ligand-binding domain (LBD) of NRs trough the nuclear receptor interaction domain (TIF2NRID) containing three highly conserved -helical LxxLL motifs (NR-boxes). The precise binding mode of this domain to RXR/RAR is not clear due to the disordered nature of TIF2. Here we present the structural characterization of TIF2NRID by integrating several experimental (NMR, SAXS, CD, SEC-MALS) and computational data. Collectively, the data are in agreement with a largely disordered protein with partially structured regions, including the NR-boxes and their flanking regions, which are evolutionary conserved. NMR and X-ray crystallographic data on TIF2NRID in complex with RXR/RAR reveal a cooperative binding of the three NR-boxes as well as an active role of their flanking regions in the interaction. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.29.317909v1?rss=1 Authors: Asthana, P., Singh, D., Pedersen, J. S., Hynönen, M. J., Sulu, R., Murthy, A. V., Laitaoja, M., Jänis, J., W. Riley, L., Venkatesan, R. Abstract: Tuberculosis (Tb), caused by Mycobacterium tuberculosis (Mtb), is responsible for more than a million deaths annually. In the latent phase of infection, Mtb uses lipids as the source of carbon and energy for its survival. The lipid molecules are transported across the cell wall via multiple transport systems. One such set of widely present and less-studied transporters is the Mammalian-cell-entry (Mce) complexes. Here, we report the properties of the substrate-binding proteins (SBPs; MceA-F) of the Mce1 and Mce4 complexes from Mtb which are responsible for the import of mycolic acid/fatty acids, and cholesterol respectively. MceA-F are composed of four domains namely, transmembrane, MCE, helical and tail domains. Our studies show that MceA-F are predominantly monomeric when purified individually and do not form homohexamers unlike the reported homologs (MlaD, PqiB and LetB) from other prokaryotes. The crystal structure of MCE domain of Mtb Mce4A (MtMce4A39-140) determined at 2.9 [A] shows the formation of an unexpected domain-swapped dimer in the crystals. Further, the purification and small-angle X-ray scattering (SAXS) analysis on MtMce1A, MtMce4A and their domains suggest that the helical domain requires hydrophobic interactions with the detergent molecules for its stability. Combining all the experimental data, we propose a heterohexameric arrangement of MtMceA-F SBPs, where the soluble MCE domain of the SBPs would remain in the periplasm with the helical domain extending to the lipid layer forming a hollow channel for the transport of lipids across the membranes. The tail domain would reach the cell surface assisting in lipid recognition and binding. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.18.291195v1?rss=1 Authors: Yui, A., Caaveiro, J. M. M., Kuroda, D., Nakakido, M., Nagatoishi, S., Goda, S., Maruno, T., Uchiyama, S., Tsumoto, K. Abstract: LI-cadherin is a member of cadherin superfamily which is a Ca2+-dependent cell adhesion protein. Its expression is observed on various types of cells in the human body such as normal small intestine and colon cells, and gastric cancer cells. Because its expression is not observed on normal gastric cells, LI-cadherin is a promising target for gastric cancer imaging. However, since the cell adhesion mechanism of LI-cadherin has remained unknown, rational design of therapeutics targeting this cadherin has been complicated. Here, we have studied the homodimerization mechanism of LI-cadherin. We report the crystal structure of the LI-cadherin EC1-4 homodimer. The EC1-4 homodimer exhibited a unique architecture different from that of other cadherins reported so far. The crystal structure also revealed that LI-cadherin possesses a noncanonical calcium ion-free linker between EC2 and EC3. We also show that LI-cadherin EC1-2 and EC3-4 have different characteristics to that of the EC1-2 domains of classical cadherins despite the sequence similarity among them. Various biochemical techniques, molecular dynamics (MD) simulations and small angle X-ray scattering (SAXS) were employed to elucidate the mechanism of homodimerization. We expect these findings will advance the generation of therapeutic molecules targeting LI-cadherin and to establish the specific role of LI-cadherin on cancer cells. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.11.293043v1?rss=1 Authors: Orioli, S., Henning Hansen, C. G., Arleth, L. Abstract: We introduce a new software, called Marbles, that employs SAXS intensities to predict the shape of membrane proteins embedded into membrane nanodiscs. To gain computational speed and efficient convergence, the strategy is based on a hybrid approach that allows one to account for the nanodisc contribution to the SAXS intensity through a semi-analytical model, while the embedded membrane protein is treated as set of beads, similarly to well known ab-initio methods. The code, implemented in C++ with a Python user interface, provides a good performance and includes the possibility to systematically treat unstructured domains. We prove the reliability and flexibility of our approach by benchmarking the code on a toy model and two proteins of very different geometry and size. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.09.290122v1?rss=1 Authors: Pinet, L., Wang, Y., Deville, C., Lescop, E., Badache, A., Bontems, F., Morellet, N., Durand, D., Guerlesquin, F., Assrir, N., van Heijenoort, C. Abstract: ErbB2 (or HER2) is a receptor tyrosine kinase overexpressed in some breast cancers, associated with poor prognosis. Treatments targeting the receptor extracellular and kinase domains have greatly improved disease outcome in the last twenty years. In parallel, the structures of these domains have been described, enabling better mechanistic understanding of the receptor function and targeted inhibition. However, ErbB2 disordered C-terminal cytoplasmic tail (CtErbB2) remains very poorly characterized in terms of structure, dynamics and detailed functional mechanism. Yet, it is where signal transduction is triggered, via phosphorylation of tyrosine residues, and carried out, via interaction with adaptor proteins. Here we report the first description of ErbB2 disordered tail at atomic resolution, using NMR and SAXS. We show that although no part of CtErbB2 has any stable secondary or tertiary structure, it has around 20% propensity for a N-terminal helix that is suspected to interact with the kinase domain, and many PPII stretches distributed all along the sequence, forming potential SH3 and WW domains binding sites. Moreover, we identified a long-range transient contact involving CtErbB2 termini. These characteristics suggest new potential mechanisms of auto-regulation and protein-protein interaction. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.09.287375v1?rss=1 Authors: Ito, S., Senoo, A., Nagatoishi, S., Ohue, M., Yamamoto, M., Tsumoto, K., Wakui, N. Abstract: Cyclic peptides, with unique structural features, have emerged as new candidates for drug discovery; their association with human serum albumin (HSA; long blood half-life), is crucial to improve drug delivery and avoid renal clearance. Here, we present the crystal structure of HSA complexed with dalbavancin, a clinically used cyclic peptide. SAXS and ITC experiments showed that the HSA-dalbavancin complex exists in a monomeric state; dalbavancin is only bound to the subdomain IA of HSA in solution. Structural analysis and MD simulation revealed that the swing of Phe70 and movement of the helix near dalbavancin were necessary for binding. The flip of Leu251 promoted the formation of the binding pocket with an induced-fit mechanism; moreover, the movement of the loop region including Glu60 increased the number of non-covalent interactions with HSA. These findings may support the development of new cyclic peptides for clinical use, particularly the elucidation of their binding mechanism to HSA. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.07.234443v1?rss=1 Authors: Chaves-Arquero, B., Martinez-Lumbreras, S., Sibille, N., Camero, S., Bernado, P., Jimenez, M. A., Zorrilla, S., Perez-Canadillas, J. M. Abstract: Yeast eIF4G1 interacts with RNA binding proteins (RBPs) like Pab1 and Pub1 affecting its function in translation initiation and stress granules formation. We present an NMR and SAXS study of the intrinsically disordered region of eIF4G1, eIF4G11-249, and its interactions with Pub1 and Pab1. The conformational ensemble of eIF4G11-249 shows an -helix within the BOX3 conserved element and a dynamic network of fuzzy {pi}-{pi} and {pi}-cation interactions involving arginine and aromatic residues. The Pab1 RRM2 domain interacts with eIF4G1 BOX3, the canonical interaction site, but also with BOX2, a conserved element of unknown function to date. In contrast, the Pub1 RRM3 domain interacts with the RNA1-1 and BOX1 regions of eIF4G1. Mixtures of Pub1, Pab1 and eIF4G1 form micrometer-size protein condensates that require the presence of the eIF4G1 BOX1 element. These homotypic interactions suggest a double key mechanism of eIF4G1 regulation, important for understanding the architecture of stress granule cores. Copy rights belong to original authors. Visit the link for more info

Seguimos celebrando nuestro primer aniversario de Si Se Puede y nos acompaña el día de hoy el Saxofonista Ernesto Saxs

Ernesto Saxs es un musico venezolano que nos ha acompañado en muchos eventos del VBC, en esta entrevista podemos comprobar que el saxo en vivo se adapta a cualquier evento y cambia el ambiente de la reunion. Esta entrevista tiene version en video tanto en Facebook como en Youtube. Visiten https://www.facebook.com/vbcradio/ para ver el video de la entrevista.

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.

This study characterizes Pur-α structurally and functionally. Pur-α is a highly conserved RNA- and DNA-binding protein involved in a multitude of cellular processes such as transcription, replication, cell cycle control, and mRNA transport. No homologous proteins with known structures are available. X-ray crystallography is often hampered by the lack of diffraction-quality protein crystals. This study demonstrates how this bottleneck was overcome by the combination of iterative use of sensitive bioinformatics tools and structure determination of a bacterial homolog. The identification of three repeat regions (PUR repeats) in eukaryotic Pur-α enabled the detection of a bacterial homolog, which corresponds to one PUR repeat. The crystal structure of Borrelia burgdorferi Pur-α was solved at 1.9 Å and was employed for precise domain boundary prediction for the Drosophila melanogaster ortholog. Therewith it became possible to obtain diffraction-quality crystals of eukaryotic Pur-α. The crystal structure of D. melanogaster Pur-α repeats I-II was solved at 2.1 Å and shares a highly conserved fold with B. burgdorferi Pur-α. One PUR repeat has an overall ββββα− topology, and two PUR repeats interact with each other to form a globular PUR domain. Small angle X-ray scattering (SAXS) analysis together with analytical size-exclusion chromatography provided evidence that dimerization of full length Pur-α requires PUR repeat III. PUR repeat III is proposed to form a PUR domain with a PUR repeat III from another Pur-α molecule. Surface envelopes calculated from SAXS data comply with this dimerization model. DNA- as well as RNA-binding properties of Pur-α were examined by filter binding assays and electrophoretic mobility shift assays. Structure-guided mutagenesis identified the β-sheets of the PUR domain as the nucleic-acid binding surface. To assess the protein-binding properties of D. melanogaster Pur-α, a yeast-two-hybrid screen was commissioned and evaluated. It confirmed the self-interaction of Pur-α and yielded Arrestin1, LaminC, Eye and Cka as putative previously unknown interaction partners.

In dieser Arbeit wurde mit Röntgenkleinwinkelstreuung (SAXS) und Fluoreszenzkorrelationsspektroskopie (FCS) die Struktur und das Phasenverhalten supramolekularer Komplexe aus Lipiden und hydrophiler DNA in unpolarem Lösungsmittel (Alkan) sowie von Komplexen aus Tensiden und hydrophoben Dendrimeren in wäßriger Umgebung untersucht. In beiden Fällen wurden Makromoleküle mit Amphiphilen komplexiert, die eine sowohl zur Oberfläche der Makromoleküle als auch zum Lösungsmittel kompatible Grenzfläche erzeugen. Weiterhin wurde im Rahmen dieser Arbeit eine Klein- und Weitwinkel Röntgenstreuanlage konzipiert und aufgebaut, die für Untersuchungen an weicher kondensierter Materie unter maximalen Fluß optimierte wurde. Der absolute Photonenfluß und die Auflösungsfunktion, sowie das Signal-Rausch-Verhalten und die zeitabhängige Speicherung des Bildplattensignals wurden bestimmt und mit der Theorie verglichen. Um eine DNA-basierte selbstorganisierte Strukturbildung in unpolaren Lösungsmitteln zu verstehen, wurden Grundlagenuntersuchungen an Lipid/DNA-Komplexen in Alkan durchgeführt und das Phasendiagramm des quaternären System aus DNA, Lipid, Wasser und Alkan bestimmt. Es wurden Lipidmischungen aus dem zwitterionischen DOPE und dem kationische DOTAP verwendet, und die Untersuchungen auf ein isoelektrisches Verhältnis zwischen DOTAP und DNA beschränkt. Das Phasendiagramm wurde als Funktion des Gewichtsanteil Phi des zwitterionischen Lipides DOPE an der Lipidgesamtmenge beschrieben. Bei einer ausreichenden Zugabe von Wasser und Alkan bilden diese zwei getrennte Phasen, wobei sich die Messungen auf die Alkanphase konzentrierten. Die Lipid/DNA-Komplexe wurden mit Röntgenkleinwinkelmessungen am Hamburger Synchrotronstrahlungslabor (HASYLAB) untersucht. Es konnte eine stabile Mesophase aus inversen zylinderartigen Lipid/DNA-Mizellen nachgewiesen werden, die bei steigendem DOPE Anteil Phi in eine Phase aus inversen sphärischen Lipid-Mizellen mit DNA-freiem Wasserkern übergeht. Zwischen beiden Phasen befindet sich ein Koexistenzbereich aus zylindrischen und sphärischen Mizellen, welcher sich zwischen Phi=72 % und Phi=82 % erstreckt. Die DNA befindet sich im Inneren der zylinderartigen inversen Lipidmizellen und ist entlang der Mizelle gestreckt. Sie wird von einer 1 nm dicken Wasserschicht von dem umgebenden Lipid getrennt. Die aus der Elektronendichteverteilung ermittelte Zusammensetzung der Lipidhülle ist gegenüber der zugegebenen Lipidzusammensetzung Phi zu einem höheren DOPE Gehalt verschoben. Aus der Interpartikelkorrelation kann eine starke Zunahme der Konzentration der Lipid/DNA-Mizellen mit steigendem Phi nachgewiesen werden. Interessanterweise ist die Struktur der zylinderartigen Lipid/DNA-Mizellen weitgehend unabhängig von der Sorte der verwendeten Alkane (Oktan, Dekan und Dodekan). Der Koexistenzbereich verschiebt sich bei Oktan in Vergleich zu Dekan und Dodekan zu einem höheren Wert. Außerdem können in Dekan für reines DOTAP (Phi=0 %) keine Komplexe festgestellt werden. Es wurde das Phasenverhalten der Lipid/DNA-Komplexe als Funktion der Wasserkonzentration bestimmt. Dies wurde exemplarisch bei einer Lipidzusammensetzung von Phi=76 % durchgeführt, bei der unter Wasserüberschuß annähernd die gesamte DNA in Alkan übergeht. Bei niedrigem Wassergehalt bilden sich in Alkan invertierte sphärische Lipidmizellen, die mit steigendem Wassergehalt anschwellen. Ab einem Wassergehalt von 163 % (Gewichtsprozent Wasser zu DNA) treten zylinderartige Lipid/DNA-Mizellen auf, deren Wassergehalt mit der zugegebenen Wassermenge bis zu einer Schichtdicke von 1 nm zunimmt. Im zweiten Teil der Arbeit wurden mit Hilfe der Fluoreszenzkorrelationsspektroskopie hydrophobe Polyphenylen-Chromophor-Dendrimere untersucht. Drei Arme des Dendrimers weisen fluoreszierende Gruppen auf, der vierte einen bioaktiven Biotinanker. Es konnte gezeigt werden, daß die Dendrimere supramolekulare Komplexe mit Tensiden formen und so in wäßrigen Medien gelöst und als multichromophorer Fluoreszenzmarker verwendet werden können. Die Komplexe zeigen bei Verwendung verschiedener Tenside unterschiedliche Strukturen. Alle weiteren Messungen wurden mit dem Tensid Tween 20 durchgeführt, das monodisperse Tensid/Dendrimer-Mizellen mit jeweils einem einzelnen Dendrimer bilden kann. Aus der Analyse der Fluoreszenzautokorrelation bei einer Dendrimerkonzentration von 50 nM erhält man zwei stark unterschiedliche Diffusionszeiten von t_D=168 µs und t_D=2470 µs, die beide über den gesamten Tensid-Konzentrationsbereich nachweisbar sind. Die schnellere Komponente aus Tensid/Dendrimer-Mizellen mit jeweils einem einzelnen Dendrimer pro Mizelle, dominiert die Autokorrelationsfunktion oberhalb einer Tensidkonzentration von 1,7e-4 M. Ihre Diffusionskonstante bleibt für alle Tensidkonzentrationen konstant und ergibt einen hydrodynamischen Radius R_H=7,1 nm. Die langsamere Komponente aus großen Aggregaten mit einer Vielzahl von Dendrimeren überwiegt unterhalb der Übergangskonzentration. Ihr hydrodynamischer Radius divergiert mit sinkender Tensidkonzentration bis hin zu einer Größe von über 20µm. Die Tensid/Dendrimer-Mizellen bleiben auch bei Verdünnung stabil. Innerhalb eines Konzentrationsbereiches der Dendrimere zwischen 10 nM und 10 M ist die gemessene Konzentration proportional zu dem Verdünnungsfaktor. Damit können die Tensid/Dendrimer-Mizellen als Fluoreszenzmarker für quantitative Fluoreszenzmessungen genutzt werden.