透過蛋白質工程學設計高ATP水解活性之OLA1突變

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研究生：	施柏仰 Shih, Po-Yang
論文名稱：	透過蛋白質工程學設計高ATP水解活性之OLA1突變 Engineering an Obg-like ATPase 1 (OLA1) Mutant with Higher Activity
指導教授：	鄭惠春 Cheng, Hui-Chun
口試委員:	孫玉珠 Sun, Yuh-Ju 邱于芯 Chiu, Yu-Hsin
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2020
畢業學年度：	108
語文別：	英文
論文頁數：	46
中文關鍵詞：	三磷酸腺苷水解活性、三磷酸鳥苷水解活性、三磷酸腺苷水解酶、三磷酸鳥苷水解酶
外文關鍵詞：	OLA1, Ras, GTPase hydrolysis, ATPase hydrolysis
相關次數：	點閱：2 下載：0
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Obg 家族蛋白是GTPase中的一大成員，並且和蛋白轉譯的執行功能有關。其中YcHF為此大家族的一分支，然而其水解ATP的效率卻比GTP高很多。在人類中與YcHF同為同源蛋白被稱為obg-like ATPase 1 (OLA1)，OLA1是由coiled-coil domain、TGS domain 和 G domain所組成的，其中G domain被認為和GTP 水解息息相關。在G domain中有五個motif在序列上是非常高度保守的，其中G1 motif 和 phosphate binding有關，G2與G3 motif則會隨著nucleotide的水解而產生結構的變化，G4和G5 motif則與鹼基的辨認及結合相關。在過去的研究中發現BARD1會藉由它的BRCT domain與OLA1產生作用並藉此調控中心粒的複製。在這裡我們想要創造人工改造OLA1，使其突變後能夠更有效率地水解ATP。未來希望這些突變的OLA1能夠與BRCT更穩定的結合，以利之後利用X-ray結晶繞射實驗了解BRCT是如何調控OLA1的。

The Obg family proteins are a large group of GTPases which are responsible for protein translation. One subfamily of this large group of proteins is YchF which has been considered as GTPase, but not ATPase, for a long time. However, it was found that human homologue of YchF could hydrolyze ATP more efficiently than GTP. Thus this protein is termed as Obg-like ATPase 1 (OLA1) [1] [2]. OLA1 contains a coil-coiled domain, TGS domain and G domain, which is important for GTP hydrolysis. In the G domain, there are five motifs, which are conserved on sequence level in all GTPases. The G1 motif (P-loop) is a loop responsible for phosphate binding. The G2 motif (switch I) and G3 motif (switch II) display nucleotide-dependent conformations. The G4 and G5 motifs are involved in base recognition and binding (Figure 4.a.). Previous studies found that BARD1 can interact with OLA1 by its BRCT domain, which is critical to DNA repair and centrosome regulation [2]. Here, we engineered OLA1 mutants having hyper ATPase-activity. This finding may be useful for biochemical and cellular studies of OLA1 and BARD1 in the future.

中文摘要    5
Abstracts    6
Chapter 1.Introduction    7
1    Breast cancer-associated susceptibility gene-1 (BRCA1)    7
2    BRCA1-associated RING domain protein (BARD1)     6
3    Ras    8
4    Human obg-like ATPase 1    8
Chapter 2.Materials and methods    10
1 Structural comparison    10
2 OLA1 mutants    10
3 Protein expression and purification    10
3.1 Purification of OLA1 WT and mutants    10
3.2 Purification of BRCT2    11
4 ATP hydrolysis assay    11
5 Circular dichroism    12
6 Isothermal titration calorimetry    12
7 Trypsin digestion    12
8 Crystallization screening    13
Chapter 3.Result    14
1 The structural superimposition of OLA1 and Ras    14
2.The chromatogram of Superdex75 revealed that the conformation of OLA1 mutants is similar.     15
3. Most mutations have opposite effects on OLA1 compared with Ras.     15
4. The secondary structure and thermostability of OLA1 N40R and G103A are similar to WT.     16
5 OLA1 N40R and Q104 were more stable in the AMPPNP state than those in the apo state.     16
6 OLA1 Q104L compared with WT had better affinity with ATP    17
7 OLA1 mutants activated by BRCT2    17
Chapter 4.Conclusion and Discussion    18
Figures    20
Reference    44


                                

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