Molecular structures of biomaterials have close relation to their functions. We are interested in how biological building blocks assemble into the structures of native biomaterials and the hierarchy of those structures. We tackled the problem mainly with X-ray diffraction experiments and developed a thorough analysis technique to assign the X-ray signals to protein secondary structures and chitin. Three different types of biomaterials were examined: vimentin fibres, squid pens, and human hair. In vimentin fibres, we found that the secondary protein structures play an important role in the strength of the fibres. In native squid pens, we found a self-similar, hierarchical structure from millimetres down to nanometres. In human hair, we compared the signals corresponding to keratin proteins, intermediate filaments, and lipids between different subjects, and found small deviations. The structures of these three biomaterials, which encompass different orders of length scales, were described both quantitatively and graphically. We hope that this work will eventually allow us to understand how and why nature builds biomaterials this way. / Thesis / Master of Science (MSc)

No description available.

No description available.

The coiled-coil motif is present in proteins from all kingdoms of life. Its structure is based on a repeating sequence of 7 amino acids with hydrophobic residues at positions 1 and 4, which folds into an alpha-helix. Two, or more, alpha-helices wind around each other based on hydrophobic interactions forming the coiled-coil. Structural variations include length, deviations from the canonical form based on the heptad repeat, as well as the orientation and number of alpha-helices. They are involved in a wide variety of cellular processes including vesicle tethering and signal transmission along their length. In order to transmit signal, the protein must be able to dynamically rearrange its structure. An outstanding example of a coiled-coil that needs to rearrange its structure to perform its function is the early endosomal tether EEA1, which has been shown to increase its flexibility upon binding to the active form of the small GTPase Rab5. That conformational change generates an entropic collapse that brings the ends of the protein closer to each other. Nevertheless, the recycling from the more flexible state to its original extended conformation was not addressed. Herein, the entropic collapse mechanism was further studied and the full EEA1 cycle between extended and flexible states described. In addition to these studies, other coiled-coil proteins were assessed to determine if they also experience a binding-induced entropic collapse. One of the strategies to investigate the entropic collapse mechanism was to compare the adhesive forces along the two alpha-helices of the EEA1 dimer in its extended and flexible conformations. To this end, an experiment was designed to unwind the dimer using optical tweezers, a force-spectroscopy method that uses a highly focused laser beam to manipulate microscopic objects. Each EEA1 monomer was attached to a distinct DNA piece using a site-specific enzymatic reaction. The DNA pieces were linked to two optically trapped micron-sized beads. And the distance between the optical traps increased to unwind the EEA1. A second strategy to investigate the entropic collapse was to evaluate EEA1 dynamics in solution using dual color fluorescence cross-correlation spectroscopy (dcFCCS). EEA1 C-termini was labeled with two different fluorophores. Fluctuations on fluorescent intensities caused by the dyes crossing a confocal volume were recorded over time. Based on an analysis of these fluctuations, a conformational change in EEA1 from semi-flexible to flexible upon addition of active Rab5 was described. This is in agreement with the previously reported entropic collapse. More importantly, EEA1 was shown to cycle between semi-flexible and flexible states by adding Rab5:GTP and waiting for the GTP to hydrolyse. To determine whether other proteins experience a binding-induced entropic collapse, coiled-coil proteins that share structural and functional similarities with EEA1 were evaluated. Rotary shadowing EM images of the target protein alone and binding with its suspected allosteric effector were compared. It was found that ELKS, a coiled-coil protein involved in vesicle trafficking, undergoes an increase in flexibility upon binding with the active form of Rab6. Thus, hinting that the entropic collapse may indeed be a general mode of action for at least a sub-group of long coiled-coil proteins. Overall, the major contributions of this thesis are to describe the full entropic collapse cycle on EEA1 and to show a second example of a coiled-coil protein experiencing a binding induced flexibility increase.:List of Figures List of Tables List of Equations List of Abbreviations 1 Introduction 1.1 EEA1 as an endosomal tether 2 Materials and Methods 2.1 Materials 2.2 Methods 2.2.1 Sub-cloning 2.2.2 Protein expression and purification 2.2.3 Protein-protein binding assays 2.2.4 Electron microscopy 2.2.5 Analysis of electron microscopy 2.2.6 Generation of DNA handles for protein-DNA conjugates 2.2.7 Adding SortaseA recognition site to EEA1 2.2.8 Protein-DNA conjugation3 2.2.9 Sample preparation for optical tweezers 2.2.10 Dual color labeling of EEA1 2.2.11 Fluorescence cross-correlation spectroscopy 2.2.12 Generation of dsDNA for dcFCCS calibration 2.2.13 RabGTPase nucleotide loading 2.2.14 Liposome preparation 2.2.15 MCBs preparation 3 Unwinding EEA1 coiled-coil domain 3.1 Introduction 3.1.1 Optical tweezers for EEA1 unwinding 3.1.2 SortaseA-catalysed ligation 3.2 Aims 3.3 Results 3.3.1 Optimization of SortaseA-catalysed ligation 3.3.2 Formation of EEA1-DNA handle conjugate 3.3.3 EEA1 unwinding experiments 3.4 Discussion 4 EEA1 entropic collapse is recyclable 4.1 Introduction 4.1.1 Advantages of dcFCCS vs FCS 4.1.2 Requirements for dcFCCS measurements 4.1.3 dcFCCS for end polymer dynamics analysis 4.2 Aims 4.3 Results 4.3.1 System preparation and dcFCCS calibration 4.3.2 Labelling of EEA1 4.3.3 Comparing FCS vs dcFCCS 4.3.4 EEA1 entropic collapse shown by dcFCCS 4.3.5 EEA1 flexibility change is recyclable 4.4 Discussion 5 Entropic collapse as a general mechanism 5.1 Introduction 5.2 Aims 5.3 Results 5.3.1 ELKS increases its flexibility upon binding active Rab6 5.3.2 p115-GM130 complex observed by rotary shadowing EM 5.4 Discussion 6 Conclusions and outlook References

In dieser Arbeit wurde eine neue Templat-gesteuerte fluorogene Peptidverknüpfung vorgestellt. Speziell modifizierte Peptidfragmente wurden durch die Bindung an ein Templatmolekül zu einer chemischen Reaktion befähigt, wodurch ein Fluoreszenzsignal erzeugt werden konnte. Das erlaubte eine Reaktionskontrolle in Echtzeit. Die fluorogene Peptidverknüpfung konnte erfolgreich mit einem Coiled-coil Peptid-Model etabliert werden. Dabei wurden Peptidthioester derart modifiziert, dass in räumlicher Nähe zur Thioestergruppe ein Fluorophor platziert wurde und die acetylierte Mercaptogruppe als Fluoreszenzlöscher agierte. Die modifizierten Thioester können nach dem Reaktionsmechanismus der nativen chemischen Peptidverknüpfung (NCL) unter der Bildung einer Amidbindung mit N-terminalen Cysteinylpeptiden reagieren. Die fluoreszenzlöschende Mercaptogruppe verlässt dabei den Peptidthioester als Nukleofug, wodurch ein fluoreszierendes Reaktionsprodukt entsteht. Die Templat-gesteuerte Durchführung dieser fluorogenen nativen chemischen Peptidverknüpfung (fNCL) erlaubte die Reaktionsdurchführung bei sehr geringen Peptidkonzentrationen. Die Synthese der benötigten fluorogenen Thioester gelang zum einem durch die Anwendung der selbstreinigenden Thioestersynthese mit einer Tandem-Entschützungs-Kupplungs-Strategie und zum Anderen mit Hilfe eines synthetisierten fluorogenen Azid-Thioesterbausteins, welches mit einem Alkin-modifizierten Peptid zur Reaktion gebracht wurde. Neben Coiled-coil Peptiden, wurden auch doppelsträngige DNA und Antikörper als Template für die fNCL eingesetzt. Die fNCL konnte zur Durchführung eines Abstandsscreenings angewendet werden. Es wurde eine Abhängigkeit zwischen der Reaktionsgeschwindigkeit und dem Abstand der Bindungsstellen im Templat für zwei reaktive Peptidbindungspartner gezeigt. Durch diese Untersuchung konnte die räumliche Abstandsgrenze zwischen zwei Bindungsstellen in einem Templatmolekül bestimmt werden, die keinen Templat-Effekt mehr beobachten lässt. / In this thesis a new template-controlled fluorogenic peptide linkage was presented. Specially modified peptide fragments were enabled to undergo a chemical reaction by binding to a template molecule, which resulted in a fluorescent signal. This allowed a reaction control in real time. The fluorogenic peptide linkage was successfully established using a coiled coil peptide model. Peptide thioesters were modified in such a way that a fluorophore was placed in close proximity to the thioester group and the acetylated mercapto group acted as fluorescence quencher. These modified thioesters can react with N-terminal cysteinyl peptides according to the reaction mechanism of the native chemical ligation (NCL) under the formation of an petide bond. The fluorescence-quenching mercapto group leaves the peptide thioester as a leaving group, resulting in a fluorescent reaction product. Template-controlled execution of this fluorogenic native chemical ligation (fNCL) allowed the reaction to be performed at very low peptide concentrations. The synthesis of the required fluorogenic thioesters was achieved on the one hand by applying a self-purifying thioester synthesis with a tandem-protective coupling strategy and on the other hand by using a synthesized fluorogenic azide thioester building block which was reacted with an alkine-modified peptide. In addition to the coiled coil peptides, double-stranded DNA and antibodies were used as templates for fNCL. Finally, the fNCL could be used to perform a distance screening. A dependence between the reaction rate and the distance between the binding sites in the template for two reactive peptide binding partners was shown. By this investigation a distance between two binding sites in a template molecule could be determined, which does not show a template effect anymore.

Das Verständnis natürlicher biologischen Materialien für die Entwicklung neuer biomimetischer Materialien ist von großem Interesse in der Chemie und den Materialwissenschaften. In vielen komplexen biomolekularen Materialien ist die Etablierung der Struktur-Funktionsbeziehungen von Proteinbausteine notwendig, um die Eigenschaften der daraus aufgebauten weichen, biologischen Materialien zu verstehen, wie z. B. die extrazelluläre Matrix. Inspiriert durch bekannte Faltungsmotive von ECM-Proteinen, wurden vereinfachte Modellpeptide entwickelt, um deren Funktion zu untersuchen oder diese als biomimetische Bausteine für synthetische Biomaterialien zu verwenden. Ziel dieser Arbeit war die Synthese von hybriden Hydrogelen, die aus einem synthetischen Polymer und ECM-inspirierten Modellpeptiden zusammengesetzt sind. Insbesondere Kollagen-mimetische Peptide und Coiled-Coil-formende Peptide wurden benutzt, um das biokompatible und hydrophile Polymer Polyethyleneglykol zu vernetzen. Dabei wurde von der Fähigkeit dieser Peptide zur dynamischen Selbstassemblierung Gebrauch gemacht. Unter Verwendung von Kollagen-mimetischen Peptiden mit langsamer Dissoziationskinetik wurden Hydrogele synthetisiert, die weichen, glasartigen Materialien mit einem gestauchten exponentiellen Relaxationsverhalten entsprechen und auch einen Alterungsprozess zeigen. Darüber hinaus wurde gezeigt, dass Netzwerkkonnektivität ein bis dato selten gebrauchter Designparameter ist, um die rheologischen Eigenschaften von Hydrogelen nach Wunsch zu kontrollieren. Die Kombination molekular einstellbarer Vernetzer mit einem Fluoreszenz-Reportersystem, welches deren Zustand auslesen kann, kann detaillierte Einblicke in das Reaktionsvermögen solcher Netzwerke auf mechanische Stimuli ermöglichen. Das Verständnis molekularer Prozesse erlaubt langfristig die Synthese von ECM-inspirierten Biomaterialien, deren Eigenschaften nach Wunsch einstellbar sind und die selbst ihren mikroskopischen und mesoskopischen Zustand anzeigen. / Understanding natural biological materials for the development of novel biomimetic materials has drawn enormous attention from the areas of chemistry and material science. In many complex biomolecular materials, establishing molecular structure-function relationships of proteins forms the basis for understanding the emerging properties of various biological soft materials, such as the extracellular matrix (ECM). Inspired by common association motifs of ECM proteins, simplified model peptides have been developed for functional studies and as biomimetic building blocks for synthetic biomaterials. The aim of this thesis was to utilize ECM-inspired and molecularly controlled model peptides for the synthesis of peptide-polymer hybrid hydrogels. Specifically, collagen-mimetic peptides (CMPs) and coiled coil (CC)-forming peptides were utilized to crosslink the biocompatible and hydrophilic polymer poly(ethylene glycol) (PEG), making use of the ability of these peptides to dynamically self-assemble. Employing CMPs with slow dissociation kinetics, hydrogels have been synthesized that resemble soft glassy materials with compressed exponential relaxation and aging. Furthermore, network connectivity has been shown to be an underutilized design parameter for tuning the rheological properties of hydrogels. Combining molecularly controlled crosslinks with a fluorescence reporter system that allows to read out crosslink status will ultimately allow for more detailed insights into the response of such networks to mechanical perturbation and thus aid the synthesis of ECM-inspired biomaterials with tunable and self-reporting properties.

Fluoreszenzmarkierungstechniken für lebende Zellen ermöglichen es Biologen, einen Blick in eine komplexe biologische Umgebung zu werfen und Informationen über ein bestimmtes Ziel in einer nahezu natürlichen Umgebung zu erhalten. Dank der konzertierten Bemühungen der wissenschaftlichen Gemeinschaft gibt es eine Fülle von kommerziell erhältlichen, genetisch kodierbaren Markern und Reportern für die Fluoreszenzmikroskopie. Allerdings gibt es nur wenige Lebendzellmethoden, die eine direkte Konjugation von Nukleinsäuren mit Proteinen erlauben, obwohl es robuste DNA-Technologien gibt, die mit Oligo-Antikörper-Konjugaten auf Zelloberflächen durchgeführt werden. Ein weiterer, oft einschränkender Aspekt der Markierung ist die Fähigkeit, Ziele selektiv zu multiplexen. In dieser Studie wurde eine Methode der Tag-Probe-Markierung entwickelt, die eine selektive, gleichzeitige Markierung von zwei verschiedenen Zielen mit zwei Peptid-Nukleinsäure-Strängen (PNA) ermöglicht. Diese Methode verwendet ein Paar von Coiled-Coil-Peptiden, um die Konjugation einer PNA-Gruppe an ein Zielprotein zu steuern, das ein Peptid-Tag exprimiert. Die Verwendung orthogonaler Coiled-Coils ermöglicht Multiplexing. Die Markierung von synthetischen Tag-Peptiden, die mittels Flüssigchromatographie analysiert wurden, hat gezeigt, dass der orthogonale duale Transfer von PNA selektiv, quantitativ und schnell ist. Die PNA-Konjugation von exemplarischen Membranrezeptoren, gefolgt von der Hybridisierung mit komplementären Fluorophor-DNAs, ermöglichte eine unkomplizierte Visualisierung von dualen Rezeptoren in lebenden Zellen. Durch den Einsatz einfacher molekularer Hilfsmittel, die die Grundlage der DNA-Nanotechnologie bilden, konnte durch die Rekrutierung mehrerer DNAs eine zunehmend hellere Markierung erreicht werden und die löschbare Oberflächenmarkierung ermöglichte eine quantitative Untersuchung der Rezeptorinternalisierung. / Live-cell fluorescent labelling techniques allow biologists to glimpse into a complex biological environment and derive information about a specific target in a near-native environment. Thanks to a concerted effort from the scientific community, a plethora of commercially available, genetically encodable tags and reporters for fluorescence microscopy exist. However, few live-cell methods allow direct conjugation of nucleic acids with proteins despite the robust DNA technologies carried out on cell surfaces using oligo-antibody conjugates. Another aspect of labelling which is often limiting is the ability to selectively multiplex targets. In this study, a method of tag–probe labelling was developed that accomplishes selective, simultaneous labelling of two distinct targets with two peptide nucleic acid (PNA) strands. The technique uses a pair of coiled-coil peptides to guide conjugation of a PNA group to a target protein expressing a peptide tag and using orthogonal coiled-coil enables multiplexing. Initially, the labelling of synthetic tag-peptides analysed by liquid chromatography revealed the orthogonal dual transfer of PNA to be selective, quantitative, and rapid. PNA conjugation of exemplar membrane receptors followed by hybridization with complementary fluorophore-DNAs achieved straightforward live-cell dual receptor visualization. Finally, using simple molecular tools that form the basis of DNA nanotechnology, recruitment of multiple DNAs facilitated progressively brighter labelling, and erasable surface labelling allowed quantitative study of receptor internalisation.

No description available.

No description available.

Master of Science / Department of Biochemistry / Qize Wei / Proper regulation of mitosis and cytokinesis is fundamentally important for all living organisms. During anaphase, antiparallel microtubules are bundled between the separating chromosomes, forming the central spindle (also called the spindle midzone), and the myosin contractile ring is assembled at the equatorial cortex. Regulators of central spindle formation and myosin contractile ring assembly are mostly restricted to the interdigitated microtubules of central spindles and they can be collectively called midzone components. It is thought that characteristic microtubule configurations during mitosis and cytokinesis are dictated by the coordinated action of microtubule-stabilizing and -destabilizing factors. Although extensive investigations have focused on understanding the roles of microtubule-bundling/stabilizing factors in controlling central spindle formation, efforts have been lacking in aiming to understand how microtubule-destabilizing factors regulate the assembly of central spindles. This dissertation describes the role of a novel microtubule-destabilizing factor termed CCDC69 (coiled-coil domain-containing protein 69) in controlling the assembly of central spindles and the recruitment of midzone components. Endogenous CCDC69 was localized to the nucleus during interphase and to the central spindle during anaphase. Exogenous expression of CCDC69 in HeLa cells destabilized microtubules and disrupted the formation of bipolar mitotic spindles. RNA interference (RNAi)-mediated knockdown of CCDC69 led to the formation of aberrant central spindles and interfered with the localization of midzone components such as aurora B kinase, protein regulator of cytokinesis 1 (PRC1), MgcRacGAP/HsCYK-4, and pololike kinase 1 (Plk1) at the central spindle. CCDC69 knockdown also decreased equatorial RhoA staining, indicating that CCDC69 deficiency can impair equatorial RhoA activation and ultimately lead to cytokinesis defects. Four coiled-coil domains were found in CCDC69 and the C terminal coiled-coil domain was required for interaction with aurora B. Disruption of aurora B function in HeLa cells by treatment with a small chemical inhibitor led to the mislocalization of CCDC69 at the central spindle. Further, vitro kinase assay showed that Plk1 could phosphorylate CCDC69. Taken together, we propose that CCDC69 acts as a scaffold and a microtubule-destabilizing factor to control the recruitment of midzone components and the assembly of central spindles.