Citrus fruits are some of the most widely consumed fruits in the world and contain significant levels of flavonoids, a category of plant secondary metabolites which control taste, color, plant defense, and overall marketability. In citrus and other plants, flavonoids are found in their glucosylated form. Glucosyltransferases (GT’s) are enzymes that add glucose to secondary metabolites like flavonoids. They make up a diverse class of enzymes ubiquitous throughout the plant and animal kingdoms. While many GT’s have been identified, they vary greatly in their structural identity, and their chemical properties make it such that only a small percentage of existing GT’s have been functionally characterized. Research on GT structure function relationships strengthens the reliability of genomic databases and makes significant contributions to the field of enzyme biotechnology. Bioenergy research and custom enzyme synthesis rely on GT structural data, making this research critical to the success of many promising current and future projects. A GT was isolated from grapefruit and was shown to glucosylate the flavonol class of flavonoids at the 3-OH position, called CP3GT. Subsequent analysis showed there are specific arrangements of amino-acids inside the catalytic cleft of CP3GT that likely account for its specificity with flavonols. These interactions are not fully understood and make CP3GT an excellent model for elucidating unique structure function relationships of a GT enzyme. X-ray crystallography is one of the best methods for structure determination that allows a 3D image of the protein in question to be resolved at the molecular level. This method has vast potential for advancing plant enzymology, yet to date only 6 plant glucosyltransferases have had their crystal structures solved. The structural similarities and complementary specificities that CP3GT shares with these crystallized GT’s make CP3GT an excellent candidate for crystallization. This research hypothesizes that there are unique structural features that give CP3GT its specificity, and that these features can be elucidated using x-ray crystallography. Wild type CP3GT and 3 recently characterized mutants are being prepared for crystallization. The crystallization of 3 CP3GT mutants in addition to wild type will compliment structure/function analysis by providing insight into how structural modifications can alter enzyme function. It is recommended that protein be in its native form for crystallization, thus a thrombin-cleavage site was inserted into WT CP3GT and 3 mutants to remove tags following purification. Some studies have suggested that the presence of tags alters enzyme activity, thus this presented the opportunity to test the effect of tags by assaying both native and tagged enzyme. Initial results showed that WT CP3GT treated with thrombin retained 70 percent activity after a 2-hour treatment at 4o C. Additional assays will be conducted to fully determine tag effects and will run concurrently with crystallization experiments

α-Synuclein (AS) is a small intrinsically disordered protein expressed in neurons and abundantly present in synapses where it is involved in regulation of synaptic vesicle-mediated protein trafficking. Misfolding of AS into amyloid fibrils is a key process in progression of Parkinson's disease (PD), the second most common neurodegenerative disorder which has no cure to date. Inhibition of AS aggregation and blocking of cell-to-cell spreading of AS fibrils is a promising strategy for PD treatment. However, rational design of inhibitors of this type remains complicated due to the lack of thorough knowledge about the mechanisms of aggregation. Therefore, the aim of this thesis was to gain deeper knowledge about AS aggregation and to apply it for developing inhibitors of AS fibrillization. In my work on the mechanisms of AS aggregation, I first determined that the concentration of AS that enables the fibril growth is an order of magnitude lower than the concentration of AS required for initial fibril formation from monomers. I explored fibril disaggregation at AS concentrations below its Kd value, and characterized AS aggregation at low micromolar concentrations. I then investigated how different modifications of AS C-terminus (namely, extensions of various sizes and charges) affect fibril growth and...

This research was designed to determine the conditions necessary to remove c-myc and 6x-His tags from a flavonol specific glucosyltransferase found in grapefruit (CP3GT) using thrombin in preparation for crystallization. X-ray crystallography of CP3GT crystals may elucidate structural features that account for flavonol specificity in some glucosyltransferase enzymes. A thrombin cleavage site was inserted into WT CP3GT and one mutant. Recombinant CP3GT was expressed in yeast and purified. Optimal conditions for thrombin digestion were explored. Digestion with 100U of thrombin for 2 hours at 4o C was optimal for removing tags from CP3GT. Storage at 4o C for 2 hours resulted in approximately 70% retention of activity. The effect of thrombin treatment on CP3GT activity was tested. Purified CP3GT protein with and without tags was tested for activity with the flavonol quercetin. Data showed no significant difference in overall activity between tagged and native protein.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第21886号 / エネ博第387号 / 新制||エネ||75(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井 孝, 教授 木下 正弘, 教授 片平 正人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM