研究生: |
周靜瑜 Ching-Yu Chou |
---|---|
論文名稱: |
利用場循環核磁共振中的弛滯技術探測蛋白質動性 Probing protein dynamics by field cycling nuclear magnetic resonance (NMR) relaxation technique |
指導教授: |
黃太煌
Huang, Tai-Huang |
學位類別: |
博士 Doctor |
系所名稱: |
物理學系 Department of Physics |
論文出版年: | 2012 |
畢業學年度: | 100 |
語文別: | 英文 |
論文頁數: | 154 |
中文關鍵詞: | 核磁共振弛秩 、高解析度場循環儀 、蛋白質動性 、自旋晶格弛秩速率 、譜密度函數 |
英文關鍵詞: | NMR relaxation, high-resolution field cycling, protein dynamics, spin-lattice relaxation rate, spectral density function |
論文種類: | 學術論文 |
相關次數: | 點閱:245 下載:0 |
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在實驗上,光譜密度函數分析是在核磁共振弛滯研究中對蛋白質動性的直接描述,而在核磁共振弛滯研究中,量測自旋晶格遲緩速率(R1)的磁場分佈則是找尋光譜密度函數的直接路徑。因此,為了探測蛋白質動性,我們設計了一個場循環裝置來量測自旋晶格遲緩速率(R1)的磁場分佈,可量測的磁場範圍為0到14.1特斯拉。這個場循環儀器可在超導磁鐵中快速地移動液態樣品,在約1公尺的距離中移動時間僅需100微秒。移動速度快且穩定,光譜有足夠的再現性。利用此儀器,我們得到了第一組蛋白質的15N-R1全磁場分佈曲線,所量測的蛋白為泛素蛋白(ubiquitin),磁場範圍為0.9-20特斯拉。出人意料地,磁場相依的15N-R1曲線無法吻合傳統的勞倫茲方程式,而產生出新的光譜密度方程式。此方程式可描述分子運動的諧波位能。本研究發現泛素蛋白的骨架動性可分為中央固定區塊和依結構兩端較動態的區塊。然而,除了泛素蛋白的動性分析,另外更重要的發現是,我們能夠從15N-R1磁場相依曲線中找出氨基酸上的諧波位能。結果顯示,位能與結構變化有著高度的相關性,當某個氨基酸具有慢速的結構變化,其位能相對會變高。此一發現可成為泛素蛋白之結構選擇結合機制的進一步證據。
Spectral density function analysis is a direct description of protein dynamics and can be extracted from NMR relaxation measurements, such as spin-lattice relaxation (R1) dispersion. Toward that goal, we have built a field cycling device for measuring R1 field dispersion curves over the field range of 0-14.1T. The device permit the shuttling of solution protein samples in regular NMR tube to be shuttled up-and-down the 1 meter superconducting magnet bore stably and reproducibly in ~ 100 ms. Using this compact field cycling device, we have obtained the first set of 15N-R1 dispersion curve of a protein, ubiquitin from 0.9 T to 20 T. Surprisingly, the field-dependent 15N-R1 curves of many residues cannot be fit with conventional Lorentzian functions and new spectral density functions based on motions subjected to harmonic potential have to be derived. The backbone dynamic information derived accordingly showed that ubiquitin contains a rigid β-strands core and mobile termini. However, the most novel finding of this thesis work is our ability to determine the harmonic potential energy for each residue from analysis of the R1 dispersion curve. The results showed that potential energy can be correlated to conformational exchange and residues having large potential energy are those exhibiting slow conformational exchange. The discovery could be a further evidence of conformational selection for ubiquitin binding mechanism.
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