Title

微粒型甲烷單加氧酶研究 -單元B之水層暴露蛋白質與銅離子作用性質

Translated Titles

A Study of the Reconstitution of Copper ions with the Partially Expressed Aqueous Exposed Domain of the Particulate Methane Monooxygenase Subunit B Protein

Authors

紀丞哲

Key Words

微粒型甲烷單加氧酶 ; 嗜甲烷菌 ; 銅離子的鍵結常數 ; Methylococcus capsulatus (Bath) ; PmoB ; Copper binding constant ; Aqueous ; Particulate Methane Monooxygenase

PublicationName

成功大學化學系學位論文

Volume or Term/Year and Month of Publication

2005年

Academic Degree Category

碩士

Advisor

俞聖法;黃得時

Content Language

繁體中文

Chinese Abstract

中 文 摘 要   Methylococcus capsulatus (Bath)為嗜甲烷菌的一種,隨著環境中銅離子濃度的高低可以調控其分別表現不同型態的微粒型甲烷單加氧酶(pMMO)及可溶型甲烷單加氧酶(sMMO)。pMMO 為膜蛋白相對於在純化上以及了解其催化機制也較為困難。過去十幾年來利用電子順磁共振光譜儀以及X光吸收光譜等實驗證實了pMMO為含有多個銅離子的蛋白質(約為12~15 個),這些銅離子可分為C-clusters 以及E-clusters 兩大類,其中E-clusters位於PmoB單元上扮演著傳遞電子至C-clusters進行催化反應的角色,因此探討銅離子與該暴露水層蛋白質間親和鍵結能力或是配位是很重要的議題。本篇論文主要是以基因工程的方法表達PmoB 單元蛋白質,並觀察在不同氧化態銅離子的環境下這些部分表達蛋白質的摺疊構形狀況以及銅離子與蛋白質間的作用關係,透過原子吸收光譜進行銅離子定量進而利用Scatchard plot 分析暴露水層蛋白質對銅離子的鍵結常數以及最大鍵結銅離子數目。由實驗結果發現銅離子扮演幫助穩定蛋白質摺疊以及與蛋白質配位兩種重要的角色,並且得知PmoB 單元暴露於水層蛋白質所含銅離子最大鍵結數目約為16.70 個。未來研究將著重於純化其它片段重組蛋白質,希望更進一步了解暴露水層蛋白質的哪些區域對於銅離子有較強的結合作用,並且將純化後之重組片段蛋白質進行蛋白質結晶進而解析其結構並偵測晶體內配位銅離子的含量。

English Abstract

Methylococcus capsulatus (Bath) is one of methanotrophic bacteria.Differential expression of either sMMO or pMMO is regulated by the concentration of copper ions available to the cells. During the past decade, the experiments from either electron paramagnetic resonance (EPR) or X-ray absorption spectroscopy conclude that pMMO is a multicopper protein. The copper ions in the pMMO were classified into C-clusters and E-clusters. The E-clusters found to be associated with water-exposed domains of the 45 kDa subunit which were responsible for channeling electrons to the C-cluster. It would be crucial to shed light on the copper binding affinity on even coordination feature in this aqueous domain of PmoB subunit to show how the electrons transfered from E-Cluster to C-cluster where the methane oxidation take place. Therefore, over-expressed PmoB subunit, a water-soluble domain of of pMMO in E.coli. system was conducted by using gene engineering technique. Thus, whether partially expressed proteins can be folded properly or the copper ions exhibt capability to sequester can be possilbly deduced. Furthermore, the copper concentration was quantitated with atomic absorption spectroscopy, and the copper binding constant was examessed by Scatchard plot. The results indicated that the copper ion played at least two important roles in both coordinating with protein and stablized the protein folding structure. The maximum number of protein binding copper is about 16.70. In the future, we will express other domains of water-exposed protein to study which sequence has stronger copper binding effect, and resolve their structures and copper coordinations via X-ray crystallography.

Topic Category 基礎與應用科學 > 化學
理學院 > 化學系
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