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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Fast photochemical oxidation of proteins coupled to mass spectrometry reveals conformational states of apurinic/apyrimidic endonuclease 1

Hernandez Quiñones, Denisse Berenice 08 July 2015 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Fast photochemical oxidation of proteins (FPOP) is an emerging footprinting method that utilizes hydroxyl radicals. The use of hydroxyl radicals create stable labeled products that can be analyzed with mass spectrometry. The advantage of FPOP over other methods is the fast acquisition of results and the small amount of sample required for analysis. Protein structure and protein- ligand interactions have been studied with FPOP. Here we evaluated (1) the reproducibility of FPOP, (2) the effect of hydrogen peroxide concentration on oxidation and (3) the use of FPOP to evaluate protein- nucleic acid interaction with Apurinic/Apurinic endonuclease 1 (APE1) protein. APE1 is a pleotropic protein that has been crystallized and studied widely. The 35641.5 Da protein has two major functional activities: DNA repair and redox function. An intact protein study of APE1 showed consistent global labeling by FPOP and a correlation between oxidation and hydrogen peroxide concentration. Furthermore, analysis of APE1 with DNA was done in hopes of probing the DNA binding site. Although the oxidation observed was not sufficient to define the complex pocket, a dramatic effect was seen in residue oxidation when DNA was added. Interestingly, the internal residues were labeled collectively in all APE1 experiments which indicates partial unfolding of the protein as previously suggested in the literature. Hence, these findings establish the use of FPOP to capture protein dynamics and provide evidence of the existence breathing dynamics of APE1.
2

Využití radikálového značení bílkovin pro strukturní biologii / Utilization of protein radical foootprinting for stuctural biology

Polák, Marek January 2020 (has links)
(In English) The reaction of highly reactive oxygen radicals with protein solvent-accessible residues can be utilized to map protein landscape. Fast photochemical oxidation of proteins (FPOP) is an MS- based technique, which utilizes highly reactive radical species to oxidize proteins and map protein surface or its interactions with their interaction partners. In this work, FPOP was employed to study protein-DNA interactions. First, a full-length of FOXO4-DBD was successfully expressed and purified. The ability of the protein to bind its DNA-response element was verified by electrophoretic and MS-based techniques, respectively. Optimal experimental conditions were achieved to oxidize the protein itself and in the presence of DNA, respectively. Oxidized samples were analyzed by bottom-up and top-down approach. In the bottom-up experiment, modification of individual residues was precisely located and quantified. Different extend of modification was observed for protein alone and in complex with DNA. To avoid experimental artifacts analyzing multiply oxidized protein, standard bottom up approach was replaced by a progressive top-down technology. Only a singly oxidized protein ion was isolated, and further fragmented by collision-induced dissociation (CID) and electron-capture dissociation (ECD),...

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