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標題: TopoisomeraseIV之ParE次單元蛋白之純化與結晶
Purification and crystallization of the ParE subunit of topoisomerase IV
作者: 林德昇
Lin, Ter-Sheng
關鍵字: ParE;拓撲異構酶;topoisomerase IV;topo IV;DNA topoisomerase;蛋白純化;蛋白結晶
出版社: 生物化學研究所
第IIA 型DNA 拓撲異構酶是細胞進行chromosome condensation 與segregation 時不可或缺的酵素,也因此是許多抗菌和抗癌藥物的目標蛋白。第IIA 型DNA拓撲異構酶反應機構的研究顯示此酵素會利用其活性中心的tyrosine residues 將兩股DNA backbones 上相隔4個鹼基對的磷酸二酯鍵切斷,再利用水解ATP所產生的能量造成蛋白結構的變化,將缺口間的鹼基對拉開形成一個大小超過 20Ǻ 的通道,以利另一雙股DNA 的通過。不過,目前已知的第IIA 型DNA 拓撲異構酶片段結構尚無法解釋ATP 之結合及水解與蛋白結構變化間之關聯,本研究目的即希望透過ParE 蛋白的晶體結構解析來了解此機制。ParE 蛋白是 bacterial topoisomerase IV (一種第IIA 型DNA 拓撲異構酶) 的次單元,其包含
有 ATPase 及 Toprim 兩個功能區。 若能比較有無ATP 存在時ParE 蛋白上此二功能區間分子介面的結構,應能對第IIA 型DNA 拓撲異構酶之反應機制提供重要的資訊。目前我們已經由親和性、疏水作用力和分子篩管柱層析法得到純化的ParE 蛋白,並且在加入ATP 類似物AMPPNP 後進行蛋白結晶。在嘗試了許多方法與結晶條件後,已獲得X-ray 繞射解析度達2.6Å 的晶體,其晶格常數為 a = 70.158Å、b =113.296Å、c =191.395Å,α=β=γ= 90°;晶系為primitive orthorhombic。然而,由於此晶體之mosaicity 太高,不利於繞射數據的收集,因此必需獲得更高品質之晶體才能有利蛋白結構的解析。目前則繼續利用 additive 方法來改善結晶條件,期待獲得更高解析度之晶體。

Type IIA DNA topoisomerases are essential enzymes for chromosome condensation and segregation, and they are the molecular targets of a variety of antimicrobial and anticancer agents. To catalyze DNA topoisomerization, a pair of active site tyrosines are used by type IIA enzymes to cleave phosphodiester bonds of the opposing strands four-base pairs apart. The two ends of the cleaved duplex DNA are then pulled apart by an ATP hydrolysis-coupled conformational change to create a gate that allows the transport of another duplex DNA.
Although X-ray crystallographic studies have provided structures for portions of type IIA topoisomerases, these structures do not give any information about the conformational change of the enzymes after ATP binding and hydrolysis. We plan to address this question by determining the crystal structure of ParE a bacterial topoisomerase IV, which contains catalytic essential ATPase and Toprim domains. Toward this goal, I purified ParE protein using a protocol that
employs affinity, hydrophobic interaction and gel-filtration chromatography. The purified protein was crystallized using MPD as precipitant in the presence of AMPPNP (a non-hydrolyzable ATP analog). After refining the crystallization
condition, we obtained a primitive orthorhombic crystal that diffracted to a resolution of 2.6Å. The unit cell parameters are a = 70.158Å, b = 113.296Å, c = 191.395Å, and α=β=γ=90°. Unfortunately, data collection was hampered by the
high mosaicity of the crystal and the long C-axis. Strategy for improving the quality of ParE crystal is currently being designed.
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