重組膜蛋白或次單元體於結構及功能上之研究

Reconstitution of Membrane Proteins or Subunits for Structural and Functional Studies

林素卿(Su-Ching Lin)

指導教授 ： 陳長謙

國立臺灣大學/理學院/化學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.00036

摘要

膜蛋白在生物系統中飾演著非常基本且重要的角色，它的主要功能為：
細胞表面物質之受質(receptors)，離子通道(ion channels)，穿透膜之運輸子
(trans-membrane transports)， 訊息傳遞者(signal transducers)，離子幫浦(ion pumps)
及能量運輸子(free energy transducers)。本論文有興趣於兩種膜蛋白系統：膜接合
孕酮受質蛋白(membrane-associated progesterone receptors；MAPRs)與微粒體甲烷
單氧酵素(particulate methane monooxygenase；pMMO)。
其中發生在膜接合孕酮受質蛋白(MAPRs)的賀爾蒙具有促使非基因性且
快速訊息傳遞之行為。我們成功地運用sticky-end 聚合酶反應將人類肝中基因轉
殖到大腸桿菌，經大量繁殖與蛋白質表現及金屬離子親和層析法(metal-affinity
chromatography)純化後，可有效地獲得兩個具有His-tag 及GST 融合的重組型膜
接合孕酮受質蛋白(pET21mapr 和pGEX_mapr)。藉由一系列光譜分析和分辨分子
大小層析法(Size Exclusion Chromatography)得知：重組膜接合孕酮受質蛋白
pET21mapr，無論於模擬生物膜的界面活性劑環境下與否，皆具有明顯α-helical 結
構；於模擬生物膜的界面活性劑環境時，由於親油性作用下促使此膜蛋白聚合分
子量大為減小，如此也可減緩膜蛋白聚集沉積。它也是一種具有高自旋氧化態鐵
離子(high spin Fe (III))鍵結的紫質鍵結蛋白(heme-binding protein)。賀爾蒙(孕酮)
的作用可以穩定pET21mapr 的結構，避免受孕酮攜帶分子-有機溶劑(如DMSO)
VI
破壞。於核磁共振2D TROSY-based 1H-15N HSQC 實驗結果可推知，孕酮可能作
用於重組膜接合孕酮受質蛋白pET21mapr 的蛋白氨酸(arginine)及氨基乙酸
(glycine) 等殘基上。
另一類頗感興趣的膜蛋白系統為微粒體甲烷單氧酵素pMMO，它由三個
次單元體組成，其中最為重要者為具有N-端及C-端水曝露區的pmob 次單元體，
它可能是電子傳遞銅離子簇(E-cluster)所在，用於提供電子給基質催化離子簇
(C-cluster)進行甲烷的氫氧化反應。我們成功地藉由分子生物技術方法而獲得一系
列重組pmoB 蛋白；並由銅離子之鍵結研究發現C-端水曝露區的pmob 次單元體
pmoBCW 蛋白可與10 個以上還原態銅離子(Cu(I))鍵結，雖然解離常數為330
μM，但有高達4.3 互助性(cooperactivity)的Hill 常數，此性質正意謂著銅離子易
於純化過程中遺漏，因此含有不足量銅離子之x-ray 繞射晶體pMMO 酵素總缺少
活性。本論文研究結果，正可瞭解還原態電子傳遞銅離子簇於pMMO 酵素催化反
應中所扮演的重要角色。

關鍵字

膜接合孕酮受質蛋白 ； 賀爾蒙 ； 紫質鍵結蛋白 ； 微粒體甲烷單氧酵素

並列摘要

Membrane proteins play significant roles in biological systems and function as
receptors, ionic channels, trans-membrane transporters, signal transducers, ion pumps
and free energy transducers, etc. In this dissertation, we focus on two membranes
proteins, membrane-associated progesterone receptors (MAPRs) and particulate
methane monooxygenase (pMMO). Steroid hormones can mediate rapid non-genomic
effects by binding to membrane-associated receptors. We have utilized the strategy of
sticky-end PCR for efficient cloning of the human liver MAPR in E. coli. BL21 (DE3)
strain, successfully purified and obtained two recombinant proteins, dubbed
pET21mapr and pGEX_mapr by using metal-affinity or/and glutathione S-transferase
affinity column. From spectroscopic studies and size-exclusion chromatography
analysis, the following results were obtained. The recombinant protein pET21mapr
exhibits α-helical structure both in the presence or absence of detergents. There is
strong hydrophobic interaction between detergent n-dodecyl-β-maltoside and
membrane-bound protein. The interaction could be used to decrease the oligomeric
state of proteins and alleviate protein aggregation. In CD studies, we show that the
hydrophobic steroid hormone progesterone protect the secondary structure of
progesterone receptor in the absence of detergent against the organic solvent DMSO,
which is typically used to deliver progesterone to the receptor. From 2D TROSY-based
VIII
1H-15N HSQC spectra, it appears that progesterone binds to the pET21mapr receptor at
residues of arginine, glycine and others. More NMR experiments are required before
the extract residues could be determined.
A second membrane protein is the pMMO enzyme from Methylococcus
capsulatus which consists of three subunits named pmoA, pmoB and pmoC. Subunit
pmoB is the largest one, and possesses two large N-terminal and C-terminal
water-exposed domains, which serve as the location of the E-clusters to provide a
reservoir of reducing equivalents for re-reduction of the C-clusters following methane
hydroxylation. In this dissertation, these recombinant pmoB proteins have to
successfully been cloned, expressed and purified. From Scatchard plots of copper
binding, we have concluded that the C-terminus water-exposed pmoBCW domain can
accommodate more than ten Cu(I) ions with dissociation constant 330μM , and that
the Cu(I) binding is highly cooperative (αH=4.3). These results clarify why the copper
ions are so readily lost during the purification process. In the x-ray structural analysis,
the preparation was devoid of ~10 copper ions, and did not possess biochemical
activity. In short, the preparation lacked the full complement of copper ions to be
functional. Thus, we have contributed to the understanding of the significance and the
role of the reduced E-cluster in the turnover of the pMMO.

並列關鍵字

MAPRs ； hormone ； heme-binding protein ； pMMO ； E-cluster and C-cluster

參考文獻

19. 張鈞威. Functional Studies of Alkane Utilizing Bacteria Pseudomonase

9. Chan, S. I.; Chen, K. H.; Yu, S. S.; Chen, C. L.; Kuo, S. S., Toward delineating the

16. Vinchurkar, M. S.; Chen, K. H.; Yu, S. S.; Kuo, S. J.; Chiu, H. C.; Chien, S. H.;

12. Zhu, Y.; Rice, C. D.; Pang, Y.; Pace, M.; Thomas, P., Cloning, expression, and

8. Yu, S. S.; Chen, K. H.; Tseng, M. Y.; Wang, Y. S.; Tseng, C. F.; Chen, Y. J.;