<p>Ankyrin repeats (ARs) are polypeptide motifs identified in thousands of proteins. Many AR proteins play a function as scaffolds in protein-protein interactions which may require specific mechanical properties. Also, a number of AR proteins have been proposed to mediate mechanotransduction in a variety of different functional settings. The folding and stability of a number of AR proteins have been studied in detail by chemical and temperature denaturation experiments, yet the mechanic of AR proteins remain largely unknown. In this dissertation, we have researched the mechanical properties of AR proteins by using protein engineering and a combination of atomic force microscopy (AFM)-based single-molecule force spectroscopy and steered molecular dynamics (SMD) simulations. Three kinds of AR proteins were investigated: NI6C (synthetic AR protein), D34 (of ankyrin-R) and gankyrin (oncoprotein). While the main focus of this research was to characterize the response of AR proteins to mechanical forces, our results extended beyond the protein nanomechanics to the understanding of protein folding mechanisms.</p> / Dissertation

Hypoxia-inducible factor (HIF) is the master regulator of genes involved in adaptation to hypoxia. The stability and transcriptional activity of HIF are regulated by post-translational hydroxylations: prolyl hydroxylation by the prolyl hydroxylase domain-containing enzymes PHD1 – 3 earmarks HIF for proteasomal degradation, whilst asparaginyl hydroxylation by factor inhibiting HIF (FIH) blocks the interaction of HIF with the transcriptional coactivators p300/CBP. The PHDs and FIH hydroxylate HIF directly from molecular oxygen and are therefore oxygen sensors. Recent literature shows that FIH also hydroxylates a number of proteins containing an ankyrin-repeat domain (ARD). Together with reports suggesting that the PHDs are involved in HIF-independent pathways, this suggests that the HIF hydroxylases may have a wide range of non-HIF targets. This thesis describes my investigations into novel substrates of the HIF hydroxylases. This work has characterized the FIH-dependent hydroxylation of the ARD-containing protein Notch1, and defined a consensus sequence for hydroxylation that corresponds to the ankyrin-repeat consensus. Using this consensus potential sites of hydroxylation in a novel ARD FIH substrate, myosin phosphatase targeting subunit 1 (MYPT1), were identified then subsequently confirmed and characterized. Notch1 competes with HIF for FIH hydroxylation. My experiments show that this occurs because Notch1 is a more efficient substrate than HIF, whilst studies on MYPT1 and other proteins indicate that competitive inhibition of FIH may be a general property of ARDs. There are more than 300 ARD proteins in the human genome, and this thesis demonstrates that FIH may hydroxylate a significant percentage of these. In addition to the analysis of ARD hydroxylation a proteomic investigation into novel PHD3 substrates has identified two candidate proteins, suggesting that the PHDs may also have multiple targets. These results have important implications for oxygen sensing, and indicate that post-translational hydroxylation is likely to be a widespread modification in cell biology.

Designade ankyrinupprepningsproteiner (DARPiner) är små, mycket stabila antikroppsmimetiska proteiner. I det här projektet användes anti-EpCAM-DARPiner tillsammans med mikrofluidik för att avgära om de kunde fånga upp HCT116-celler mer effektivt än anti-EpCAM-antikroppar. Ytorna på insidan av mikroffluidikkanaler förändrades genom bindning av N-γ-maleimidobutyryl-oxysuccinimidester (GMBS) och merkaptopropyltrietoxysilan (MPTES) för anti-EpCAM-antikroppar och GMBS och (3-aminopropyl)trietoxysilan (APTES) för DARPiner. Båda kanaltyperna testades genom inflöde av cancerceller och helblod blandat med cancerceller. Ingen effektiv och konsekvent celluppfångst åstadkoms trots att det visades att antikropparna och DARPinerna kunde binda till cellerna direkt och att test med fluorescenta DARPiner och antikroppar visade att ytförändringskemin var fungerande. Slutsatsen blev att de mest troliga orsakerna till misslyckandena var att ytförändringskemin påverkade proteinernas bindningsförmåga negativt eller att proteinerna bands till kanalernas yta i fel riktning. DARPiner är fortfarande intressanta för tillämpningar inom mikrofluidik, men vidare förbättring av det experimentella protokollet behövs. / Designed ankyrin repeat proteins (DARPins) are small and highly stable antibody mimetics. In this project, anti-EpCAM DARPins were used in conjunction with microfluidics to determine if they could capture HCT116 cells more effectively than anti-EpCAM antibodies. The inside surfaces of microfluidic chips were modified using N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS) and mercaptopropyltriethoxysilane (MPTES) for anti-EpCAM antibodies, and surface modifications for anti-EpCAM DARPins were made using GMBS and (3-aminopropyl)triethoxysilane (APTES). Both chip types were tested using cancer cells and whole blood mixed with cancer cells. No effective and consistent cell capture was achieved, despite the antibodies and DARPins being shown to be able to bind to the cells directly and tests with fluorescently labelled DARPins and antibodies showing that the surface modification chemistry used was functional. It was concluded that the most likely causes of the failures were surface modifications interfering with the binding ability of the proteins, or improper orientation of the bound proteins. The DARPin remains a protein of interest for microfluidic applications, but further changes and optimisation of the experimental protocol is necessary.