台灣中度嗜熱菌MtaLonA1蛋白酶之純化與其蛋白質 晶體之分析

Purification and crystallization of MtaLonA1 from Meiothermus taiwanensis

楊政諭(Cheng-Yui Yang)

指導教授 ： 張崇毅

國立臺灣大學/生命科學院/生化科學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10241

摘要

在細胞中蛋白質的合成與降解是處於一個動態的平衡狀態，有新的蛋白質合成也有失去功能與無法正常摺疊之蛋白被降解。兩者之間的平衡一但被破壞將導致細胞的環境失去恆定性，導致細胞因此產生警示性的反應而引發細胞凋亡以及細胞癌化的可能。
　　在眾多調控細胞蛋白質和成與降解的機制中，有一群被稱為AAA+ protease family的蛋白脢。它們共有的特徵就是在其中具有ATPase的AAA+ module可以跟ATP結合與水解，因此產生蛋白質構形改變與用此能量來unfolding目標蛋白。它們通常是辨認目標蛋白於不正常摺疊時所露出的特定序列，通常以”degron”稱呼之。在細胞中它們如同品管人員一般，進行protein quality control以及決定特定蛋白在細胞中的表現水平。在AAA+ protease family之中有一類蛋白脢名為‘'Lon protease”，依照序列比對又可再細分為LonA、LonB與LonC三類。LonA由N端之N domain以及接下來的AAA+ domain與protease domain組成；LonB與LonC較為相似，LonB相較於LonA少了N domain但AAA+ domain有多出穿膜區段，LonC其AAA+ domain則是去活性的，在AAA+ domain上有延伸出一段 coiled-coil domain。其中LonA不論是原核與真核生物的胞器都可以發現它的存在。於真核生物中它存在於粒線體、葉綠體、過氧化體這些胞器，因此它在維持細胞能量代謝以及氧化壓力水平扮演著重要的角色。
　 到目前關於LonA完整的結構尚未解出，目前解出的多為獨立功能域的結構。因此在本篇研究嘗試以Meiothermus taiwanensis之MtaLonA1蛋白用於長晶，希望能拼湊出MtaLonA1蛋白完整的結構。雖然我們的確有長出MtaLonA1蛋白全長晶體，X-ray繞射之最佳解析度介於3~3.5A，但是晶格某一方向的軸異常的長(468.38A)，此晶體繞射品質尚在改進中。目前另採取其他策略把全長蛋白分成兩個蛋白用於長晶，其一為涵蓋N端至AAA+ domain之α/β sub domain；另一為去除N domain的蛋白。

關鍵字

AAA + protease ； Lon protease ； degron ； protein quality control ； X-ray 繞射 ； Meiothermus taiwanensis ； MtaLonA1

並列摘要

Lon proteases belong to a family of AAA+ (ATPases associated with diverse cellular activities) proteases, which perform protein quality control in bacteria and eukaryotic organisms. The Lon protease family has been divided into three subfamilies, LonA、LonB and LonC, based on sequence homology and domain organization. LonA proteases are composed of a large N-terminal domain, a central ATPase domain, and a C-terminal protease domain. LonB proteases lacked the N-terminal domain of Lon A; however, they contain transmembrane regions inserted within the ATPase domain. LonC lacked ATPase activity and the transmembrane regions in LonB was replaced by the coiled-coil domains. So far the structure of full-length LonA has remained elusive, which has limited the understanding about the molecular mechanisms for substrate recognition and processing.
Here we describe expression, purification, and crystallization of MtaLonA1 from Meiothermus taiwanensis(Mta). The largest crystals grew in 4~6 day at 22 °C to the size of 0.3 × 0.2 × 0.05 mm, with diffraction resolution reaching 3~ 3.5 A. But one of the axis in the unit cell was long (468.38A), and the data set could not be processed due to high mosaicity.
As an alternative, we have made two truncated MtaLonA1 proteins for crystal growth. Mta_NAAA contains the N-terminal to the AAA + the domain of the α/β sub domain；and Mta_AAAP which contains the AAA+ domain and protease domain. Structures of these constructs may be used to assemble a full-length model of MtaLonA1 by superimposing their overlapping AAA domains. The expression, purification, and crystallization of these constructs are described.

並列關鍵字

AAA + protease ； Lon protease ； degron ； protein quality control ； X-ray diffraction ； Meiothermus taiwanensis ； MtaLonA1

參考文獻

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