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Chlorophyll spielt eine unabdingbare Rolle für die lichtabhängige Reaktion der Photosynthese. Die adäquate Versorgung mit Chlorophyll wird dabei durch die Tetrapyrrolbiosynthese (TBS) gewährleistet. In den letzten Jahrzehnten wurde eine Vielzahl von Proteinen identifiziert, welche an der Anpassung der TBS an wechselnde (a)biotische Wachstumsbedingungen der Pflanze beteiligt sind. Allerdings konnte bislang nicht zweifelsfrei geklärt werden, wie die TBS mit der Integration von Chlorophyllen in die Photosysteme koordiniert wird. Vor einigen Jahren wurde ein Interaktionspartner der Protochlorophyllid-Oxidoreduktase (POR) in Synechocystis identifiziert, welcher als potenzieller Faktor dieser Koordination in Frage kommt. Das POR-INTERACTING TPR-Protein (Pitt) stabilisiert POR an der Thylakoidmembran und interagiert auch mit dem Vorstufenprotein des D1. Pitt gehört zur Familie der tetratricopeptide repeat (TPR) Proteine, deren Vertreter vorrangig für die Vermittlung von Protein-Protein-Interaktionen zuständig sind. Aus diesem Grund war, neben der Identifikation des potenziellen Pitt-Homologs im Modelorganismus Arabidopsis thaliana, die Analyse von anderen Vertretern dieser Proteinklasse ein vielversprechender Ansatz bei der Identifikation von weiteren Regulatoren der TBS oder Photosynthese. Von den fünf ausgewählten TPR-Proteinen aus Arabidopsis thaliana mit einer hohen Sequenzähnlichkeit zu Pitt waren vier in der Lage, physisch mit POR zu interagieren. Von diesen vier Kandidaten ist das durch das Gen At1g78915 kodierte, membranintegrale TPR-Protein (TPR1) der beste Kandidat des putativen Pitt-Homologs in Arabidopsis. Vergleichbar zu Pitt interagiert TPR1 mit POR und stabilisiert das Enzym an den plastidären Membranen. Die Stabilisierung von POR durch TPR1 spielt eine entscheidende Rolle während der Etiolierung und Ergrünung von Keimlingen. Darüber hinaus steht TPR1 im Zusammenhang mit der schnellen Inaktivierung der 5-Aminolävulinsäuresynthese. / Chlorophyll plays an indispensable role in the light reaction of the photosynthesis. The adequate supply of chlorophyll is ensured by tetrapyrrole biosynthesis (TBS). Within the last decades, multiple proteins were identified, which are involved in adjusting the TBS-pathway to changing (a)biotic plant growth conditions. Nevertheless, it is not fully understood how the TBS-pathway is coordinated parallel to the assembly of the photosystems and the integration of chlorophylls into the pigment-binding subunits of the photosystems. Several years ago, an interaction partner of the protochlorophyllide-oxidoreductase (POR) was identified in Synechocystis which was proposed to be involved in the coordination of these mechanisms. The POR-INTERACTING TPR-Protein (Pitt) binds and stabilizes POR at the thylakoid membranes and interacts with the precursor protein of D1. Therefore, Pitt could facilitate the incorporation of chlorophylls into the plastid-encoded nascent photosynthetic subunits. Pitt belongs to the tetratricopeptide repeat (TPR) protein family, whose members mediate protein-protein-interactions. Besides the identification of the potential Pitt-homolog in the model organism Arabidopsis thaliana, analysis of additional members of the TPR-protein superfamily was a promising approach for the identification of further posttranslational regulators of TBS and photosynthesis. Five Arabidopsis thaliana TPR-proteins with a high sequence similarity to Pitt were selected. Four of those proteins are able to interact physically with POR. Among them, the TPR-protein encoded by the gene At1g78915 (TPR1) was the best candidate to represent a putative Pitt homolog in Arabidopsis. Similar to Pitt, TPR1 is a plastid-localized integral membrane protein, which interacts with POR at the thylakoid membranes. The stabilizing effect of TPR1 on POR is especially needed during etioliation and greening. Additionally, TPR1 is required for a inactivation of the 5'-aminolevulinic acid synthesis.

STRAP (Stress responsive activator of p300) is a 440 amino acid protein, predicted to have 6 TPR (Tetra-Tri-Co-Peptide Repeats) motifs, known to mediate protein-protein interactions. STRAP has been shown to form a complex with proteins p300 and JMY (Junctional Mediatory Protein), and is implicated in the DNA damage, heat shock response pathway, regulation of the Glucocorticoid receptor and in the function of p53.The aims of this project were to clone, express and purify full length and truncated human STRAP (hSTRAP) variants in high quantities. Full length and shorter hSTRAP fragments, which contain different combinations of the predicted TPR motifs and hence cover different regions, would be then structurally characterised by various structural and biophysical experiments. Another important aim was to identify interacting partners of hSTRAP in breast cancer and to map the position of their interaction sites to different parts of the protein. To this direction GST- and His- tagged full length hSTRAP, as well as His- tagged truncated hSTRAP protein variants have been successfully cloned, expressed and purified. Independent and reproducible biochemical pull-down assays have been carried out in MCF7 breast cancer cells, followed by mass spectrometry-based proteomics analysis which identified 25 hSTRAP-interacting partners from various signaling pathways such as regulation of the actin cytoskeleton and translation. In addition, crystallization trials were carried out using pure His-hSTRAP(1-440) protein, which were unfortunately un-successful. Various hSTRAP protein variants have been characterized by CD, showing that hSTRAP(1-150), His-hSTRAP(1-440), hSTRAP(1-219), hSTRAP(151-284) and hSTRAP(285-440) comprise of alpha and β structures, but the hSTRAP protein variants show no clear cooperative unfolding transitions, suggestive of molten globule states. NMR on hSTRAP(1-219), hSTRAP(1-150) and hSTRAP(151-284) have shown these proteins are not folded at a tertiary structure level. We conclude that a protocol has been established to clone, express and purify various hSTRAP variants and the thermal and secondary structure characteristics of each have been determined, although the 3D structure could not be solved. Pull-down assays followed by proteomic analysis have shown that hSTRAP is implicated in many aspects of cellular regulation.

<p>NatA binds inositol hexakisphosphate and other ligands, and exhibits conformational flexibility dependent on the ligand bound.</p> / Dissertation

This thesis focuses on the study of TTL protein family in the model species Arabidopsis thaliana. It sums up available published data on this so far poorly studied genes and presents newly gained experimental data. Main emphasis is given to TTLs cellular localization in possible relationship to their physiological function. Presented thesis comprises of in silico analysis of TTL proteins, but also the creation and microscopy analysis of TTL-mRuby2 and TTL-GFP fusion proteins in transient and permanent transformants. Powered by TCPDF (www.tcpdf.org)

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