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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生物化學研究所 > 依資料類型分類 > 碩博士論文 >  BacteriophageT2之第二型DNA拓撲異構酵素gene39蛋白次單元的純化及結晶暨Corynebacteriumglutamicum中Prephenatedehydratase的純化及結晶

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/97528

標題: BacteriophageT2之第二型DNA拓撲異構酵素gene39蛋白次單元的純化及結晶暨Corynebacteriumglutamicum中Prephenatedehydratase的純化及結晶
Purification and crystallization of the gene 39 protein subunit of bacteriophage T2 type II DNA topoisomerase & Purification and crystallization of the Prephenate dehydratase of Corynebacterium glutamicum
作者: 林其傳
Lin, Chi-Chuan
Contributors: 詹迺立
Nei-Li Chan
國立中興大學
關鍵字: 第二型DNA拓撲異構酶;gene 39蛋白次單元
type II topoisomerase;gene 39 protein subunit;Corynebacterium glutamicum;Prephenate dehydratase
日期: 2003
Issue Date: 2012-08-31 16:53:17 (UTC+8)
Publisher: 生物化學研究所
摘要: 摘要 (section 1)
Phage T2 的第二型DNA拓撲異構酶 ﹝DNA topoisomerase Π﹞可藉由ATP的結合與水解來改變DNA的拓撲構形。此酵素是由兩個gene 39 蛋白次單元與兩個gene 52 蛋白次單元組成的tetramer,其中gene 52 蛋白為DNA cleavage/rejoin domain,而gene 39蛋白則包含Toprim domain及ATPase domain。先前的研究顯示:topoΠ在催化反應時DNA cleavage/rejoin domain會先結合並切開第一條雙股DNA﹝G-segment﹞,接著第二條DNA duplex﹝T-segment﹞可在第一個ATP水解及Toprim domain的幫助下通過酵素和第一條DNA所形成的閘口﹝gate﹞;最後酵素將第二個ATP水解並還原G-segment上的缺口以進行下一個循環的反應。雖然目前已知yeast topoΠ之Toprim domain與E. coli gyrase的ATPase domain的X光晶體結構,但尚未了解Toprim domain如何傳遞ATP結合與水解的訊息來促使酵素完成反應。因此本實驗即是要探討ATP的結合與水解如調控 Toprim 與 ATPase domains間的蛋白質交互作用;在本實驗中,我以硫銨沉澱法、離子交換樹脂管柱與分子篩管柱純化gene 39蛋白次單元以進行結晶,並使用不會被水解的ATP類似物─AMP-PNP來使gene 39蛋白停留在ATP-bound構形;在嘗試許多結晶條件後,我成功的以2.0 M Ammonium Sulfate、0.01 M Magnesium Sulfate與0.05 M Sodium Cacodylate在pH 6.5、21℃下得到此蛋白的微小晶體;為了得到較大的晶體以進行後續的X光繞射實驗,曾以上述條件為基礎加以微調並加入各種不同的添加物,結果發現:在以0.01 M氯化釔作為添加物,以及2.22 M Ammonium Sulfate、0.03 M Magnesium Sulfate與0.05 M Sodium Cacodylate在pH 6.5、21℃時可以得到更好的晶體,但此晶體的品質目前仍不足以進行X光繞射實驗。
摘要 (section 2)
Corynebacterium glutamicum中的prephenate dehydratase是參與phenylalanine生合成的重要酵素;當芳香性胺基酸的前趨物chorismate經由chorismate mutase作用後形成prephenate後即可藉由prephenate dehydratase的作用產生phenylpyruvate,之後再以aminotransferase進行轉胺作用後便可以生合成phenylalanine。
phenylalanine的生合成作用是受到許多調控的,phenylalanine本身便可以feedback inhibition的作用抑制chorismate mutase與prephenate dehydratase以及一些上游的生合成反應,此外prephenate dehydratase也可受其他兩個芳香性胺基酸─tryptophan的feedback inhibition與tyrosine的activation。
基於胺基酸在日常生活中的使用日漸廣泛,諸如食品添加物、醫藥化學用品中胺基酸的使用量亦逐年增加,因此如何提高胺基酸的工業生產量是一重要的課題。生合成phenlyalanine中的主要酵素─prephenate dehydratase的生化特性分析目前已由中興大學分生所許世光學長進行中。本實驗的目的即是要以結構生物學的角度提供prephenate dehydratase立體結構上的資訊,配合此酵素在生化功能特性上的實驗分析,了解此酵素在催化與回饋抑制上的機制。
目前已可在0.2 M KCl,0.1 M Mg Acetate,0.05 M Na Cacodylate pH 6.8,4% PEG 8000的條件下於21℃中得到蛋白質晶體,但此蛋白質晶體是呈現二維的生長方式,厚度尚不足以進行X-ray繞射,在嘗試修改許多結晶條件後仍無法改善此現象,因此在接下來的實驗中我們將要嘗試重新設計clone,切除此蛋白質N端或C端不需要的區域,並嘗試與抑制物m-fluorophenylalanine共同結晶,希望能藉此方法改善晶體的品質,進行X-ray繞射實驗。
Abstract
The DNA topoisomerase Π of phage T2 changes DNA topology by coupling the strand passage reaction to the binding and hydrolysis of ATP. This tetrameric enzyme is consisted of four subunits of two kinds, gene 52 protein and gene 39 protein. The gene 52 protein is the DNA cleavage/rejoin domain while the gene 39 protein contains Toprim domain and ATPase domain. Results from earlier studies indicate that a cascade of conformational changes and dynamic domain interactions are involved during catalysis. Although the X-ray crystal structures of the yeast Toprim domain and the ATPase domain of E. coli gyrase are known, there is no information regarding how the signal of ATP binding and hydrolysis is relayed by the Toprim domain to the cleavage/rejoin domain complete the strand passage reaction. Therefore, I would like to characterize the interface between the Toprim and ATPase domains and investigate how this interface responds to ATP binding and hydrolysis. In this experiment, the gene 39 protein subunit used for crystallization was purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatograph. Replacement of ATP by the non-hydrolysable ATP analogue─AMP-PNP results in the formation of gene 39 protein dimer. After many crystallization conditions were tested, it was found that crystallization can be achieved by mixing protein solution in an 1:1 ratio with 2 fold diluted well solution containing 2.0 M ammonium sulfate, 0.01 M magnesium sulfate and 0.05 M sodium cacodylate pH 6.5. Crystals grew within 72 hours at 21 ℃. Since this crystal is too small for X-ray diffraction studies, the crystallization condition was refined and additive screen was performed. Slightly bigger crystals were obtained at 21 ℃ when protein solution was mixed 1:1 with 2 fold dilutied well solution containing 2.22 M ammonium sulfate, 0.03 M magnesium sulfate, 0.05 M sodium cacodylate pH 6.5 using 0.01 M yttrium chloride hexahydrate as additive. But the crystal quality is still not good enough for collecting X-ray diffraction data.
Appears in Collections:[依資料類型分類] 碩博士論文

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