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577.112 --- 577.112 Proteins --- Proteins --- Theses

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Proteins can be considered as nature's most diverse building blocks which are involved in the various essential processes that constitute life. However, in the field of synthetic biochemistry and bio-nanotechnology, DNA has been the most successful building block so far to create a variety of shapes and applications, such as diagnosis and drug delivery systems. The preference of DNA over protein building blocks arose due to the fact that the design of DNA complexes is better understood, easier to design, and inexpensive. Nevertheless, protein building blocks started to gain more interest in the last few decades. Not only due to the structural and catalytic versatility protein building blocks yield, but also due to the improved understanding of protein structures and functions, the increased computational power and accessibility, and the prior successes which stimulated the research. This includes the de novo design of novel proteins, folds, and functions. Henceforth, protein building blocks can be adapted for diverse functions by combining specific protein domains and subsequently functionalising them. These protein building blocks are then suitable for various applications due to their diverse shapes and functions that can be obtained, thereby finding applications within different fields such as therapeutics, bio-electronics, and bio-materials. Nevertheless, the design of proteins and protein building blocks remains challenging due to the lack of complete understanding how proteins fold into stable structures and the general control over the position and flexibility of sub-domains.The work described here mainly focuses on the validation of a previous designed protein named Pizza. Pizza is a circular, symmetric protein with a β-propeller fold. The protein was designed by computationary reverse engineering the natural evolution of a pseudo-symmetric β-propeller to obtain a fully symmetric protein, which was named Pizza. Furthermore, Pizza derivatives demonstrated to be capable of catalysis as well as metal coordination. Here, we further explore the robustness and specificity of Pizza as a fusion-based protein building block for metal-induced tubular formation. Finally, a new, eight-fold symmetric building block is designed and validated in a similar way as Pizza to complement our symmetric protein building blocks.The first chapter starts with a general introduction of protein engineering. It highlights the rational designs starting from α-4 and Felix and continues to the latest development of computational techniques. Thereafter the focuses switches to the design of repeat proteins, more specifically the globular repeat proteins such as TIM-barrel proteins and β-propeller proteins. Finally, the applicability of artificial symmetric proteins is described in the last section of this chapter. The second chapter describes the specific goals of this manuscript.The third chapter is based on a publication and describes the design of small molecular assemblies following a fusion based approach with Pizza. Fusion proteins were designed with minimal computational effort to assemble as compact hexameric structures. Subsequently, the designs were structurally validated based on their assembly, molecular weight, and structure, thereby demonstrating the robustness of Pizza and their potential as protein scaffolds. Additionally, each complex has two Pizza proteins displayed at opposite sides of the assembly. The best scoring protein was further functionalised with an eGFP and validated accordingly. Following this development, tubular formation could be obtained via the preserved metal coordination interfaces at opposite sides of the assemblyThe fourth chapter analyses the second requirement for tubular formation, namely the metal coordination and specificity of Pizza proteins. A model was developed via the comparison of different Pizza mutants describing the transition from the Pizza coordinated cadmium nanocrystal to the observed cadmium nanoring. Furthermore, the multi-specificity of Pizza proteins was demonstrated via the crystallographic analysis of Pizza proteins in the presence of different metal ions. However, this could not be further confirmed via other techniques due to the metal induced formation of aggregates. Although these results indicate that Pizza proteins can not be used for tubular formation, they highlight the requirement of robust scaffolds to accommodate the specific coordination geometries of transition metals, if used for metal binding applications.The fifth chapter focuses on the design and validation of a new eight-fold symmetric β-propeller, named Bagel. Subsequently, Bagel was expressed and characterised as proteins containing two to nine blades with or without a Velcro closure. The thermal stability of each protein was determined, as well as their assembly. Similarly to the previous chapter, the metal coordination ability was assessed in the presence of different metal ions, leading to the dimerisation of Bagel. Lastly, preliminary results were obtained, indicating the robustness of Bagel proteins and the possibility of utilising them as a scaffold for fusion-based assemblies or for metal-induced tubular assembly.Conclusions and future perspectives are given in the final chapter of this manuscript. The appendices contain supporting information concerning sequences, characterisation, and the health, safety, and environment section.

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We successfully employed computational methods to discover a new AR-targeted therapy for the treatment of prostate cancer. Prostate cancer is the second most common cancer and affects millions of men each year worldwide. Targeting AR LBD dimerization could discover novel inhibitors that might overcome resistance mechanisms that occur in the current therapy. Here, we successfully developed the first-generation AR LBD dimerization inhibitors using structure-based drug design. The drug design process consists of two parts: (i) computationally investigating the AR LBD homodimeric structure and its druggability, (ii) performing a virtual screening to discover novel AR LBD dimerization inhibitors. Our findings indicated that AR LBD dimerization might be best characterized by an intermediate state between the crystal structure of the AR LBD homodimer and the GR-like AR LBD homodimer. A dynamic druggable binding site was found at the dimer interface of the AR LBD homodimer. Thus, this binding site can be a therapeutic platform to discover novel inhibitors for AR LBD dimerization. In the virtual screening, a database of 1M compounds was screen by combining pharmacophore- and molecular docking-based methods. The screening discovered 29 compounds purchased for the biological assays. 5 potential hits were confirmed and belong to three scaffold-based groups. To rapidly explore structure-activity relationships (SAR) around the hits, their close analogs were found by SAR by catalog and tested in the biological assays. Two other drug design projects were carried out in parallel. By performing homology modeling, pharmacophore search, and molecular docking; we made the CCR8 model and predicted the binding poses of the naphthalene-sulfonamide-based CCR8 antagonists. These insights were used to explain the SAR and also to design improved antagonists. In addition, we also investigated the binding poses of novel betulonic-acid-based H229E nsp15 inhibitors using molecular docking. The predicted results may also include SAR, the explain its specificity and aid in the development of pan-corona inhibitors. We can conclude that CADD is a powerful tool to develope novel small molecule-based therapies and to explain the activity of small molecules to target proteins. Here, we identified the new AR-tergeted therapy for treating protate cancer and predicted the binding of the novel CCR8 and H229E nsp15 inhibitors.

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Thanks to their impressive number and structural variability, proteins accomplish an amazing number of structural, catalytic and immunity functions in Nature. Additionally, their high specificity and affinity for their substrates make them a very promising option for applications in technology. Protein-based materials are one way of exploiting protein’s unique capabilities with a great potential for biotechnology applications including, but not limited to, sensing, diagnostics and catalysis. These materials can be formed by the bottom-up self-assembly of protein monomers to generate ordered and extended two-dimensional or three-dimensional protein networks. Different kinds of intermolecular interactions can be exploited to direct the protein self-assembly, but one of the key characteristics of the starting protein monomers is their symmetry. SAKe (Self-Assembling Kelch) proteins, with their symmetrical structure might represent a promising candidate for the formation of two-dimensional protein arrays. Additionally, they present a central cavity and loops that could be further functionalised. Metal-binding sites can be added to the amino acid sequence of the SAKe proteins and metal ions in solution can be used to direct protein-protein interactions and their ordered self-assembly on surfaces to form two-dimensional protein arrays. In this work, the self-assembly behaviour at the solid-liquid interface of three SAKe proteins, different from each other in number of subunits and number of metal-binding sites, was evaluated by in-liquid Amplitude-Modulated Atomic Force Microscopy (AM-AFM). Two materials were used as substrates: mica, a hydrophilic material and Highly Oriented Pyrolytic Graphite (HOPG), a hydrophobic one. A Cu(NO3)2 solution was added to the protein solution as source of Cu(II) ions; different protein concentrations were tested while keeping the protein-to-metal ratio constant. Additionally, in some cases a different pH was tested and experiments consisting of the sequential deposition of protein solution and Cu(NO3)2 were also performed. In-liquid AM-AFM revealed significant differences in the self-assembly behaviour of the different SAKe proteins. The influence of the substrate also played a very significant role, with very different results observed for the same solutions imaged on the two different surfaces, highlighting how the surface-protein interactions can have an influence on the protein-protein interactions which ultimately affects protein self-assembly on surface. Protein concentration was also shown to be a relevant parameter in controlling not only the surface coverage but also affecting the self-assembly behaviour of proteins on surfaces. Lastly, particular on HOPG, the mechanical force applied by the oscillating AFM tip was shown to affect protein organization. In conclusion, in this study in-liquid AM-AFM was proved effective not only for the imaging of protein self-assemblies on two different substrates but also as a mean of influencing their organization with a high degree of spatial control. The symmetrical SAKe proteins proved to be a promising starting point for the formation of ordered, metal-mediated protein self-assembly, which can be tuned both by modulating their concentration on solution and by careful choice of substrate. Additionally, the possibility of further functionalising their cavity or their loops favors them as candidate building blocks for protein-based 2D materials.