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標題: | 探討鋅離子及銅離子對於與阿茲海默症N-端突變家族性遺傳相關乙型類澱粉胜肽之鍵結性質 Investigating the binding properties of Zn2+ and Cu2+ to beta-amyloid peptide with familial N-terminal mutations |
作者: | Yi-Fan Liao 廖宜凡 |
指導教授: | 王勝仕(Sheng-Shih Wang) |
關鍵字: | β-類澱粉蛋白,金屬離子,光譜法,滴定曲線, β-amyloid,metal ions,spectrometry,titration curve, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 目前阿茲海默症的研究中指出,β-類澱粉蛋白(β-amyloid peptide)是造成阿茲海默症最主要的致病原因,類澱粉前驅蛋白(amyloid precursor protein, APP)經由兩種蛋白酵素水解以後產生β-類澱粉蛋白,而此種蛋白會在腦中造成不正常聚集(aggregation),形成具有神經毒性(neurotoxicity)的腦部斑塊(plaque),進一步造成神經細胞受損,此種學說稱之為類澱粉蛋白致病假說(amyloid cascade hypothesis)。
依照發生時間的不同,阿茲海默症又可被分為家族性(familial)及偶發性(sporadic)兩種,前者往往是因為遺傳突變基因導致早發性(early-onset)阿茲海默症的發生,且研究發現此類型患者其β-類澱粉蛋白聚集之機制往往與其他人有顯著的不同。 此外,對阿茲海默症患者的腦部研究後發現,在其斑塊中鋅、銅、鐵、鋁等金屬離子也發生堆積的現象,因此金屬離子被懷疑可能是誘導β-類澱粉蛋白產生聚集的可能因素之一。 本實驗為研究鋅、銅離子與原生型、及各種突變型β-類澱粉蛋白之間作用力,以試著找出金屬離子與β-類澱粉蛋白之鍵結結構(coordination),首先試著生產與純化15N標記的原生型及突變型β-類澱粉蛋白以進行1H-15N HSQC之實驗,然而由於TEV蛋白脢之切割產率過差,且最後HPLC之純化無法將帶有histag之融合蛋白有效分離,此實驗流程有待進一步之研究與改良,若假設蛋白脢之切割率為100%則約可獲得0.4mg/L之產率。 接下來試著以金屬離子對原生型及突變型的合成胜肽(synthetic peptide)進行滴定,並以各種光學法及ITC來比較其差異性。自身螢光的結果中顯示,在與鋅離子結合時,H6A及H6R兩種突變型與其他的突變型有明顯的差異。Bis-ANS螢光結果則可發現,當銅離子與β-類澱粉蛋白結合時,疏水性裸露區域減少,鋅離子與β-類澱粉蛋白結合時疏水性裸露區域則增加,可見鋅、銅離子與β-類澱粉蛋白之結合機制必定有所不同。而H6A、H6R與鋅離子結合之Bis-ANS光譜也可發現與其他類型蛋白有所不同。圓二色光譜之研究發現,β-類澱粉蛋白單體在利用氫氧化鈉去摺疊法製備時其二級結構為不規則形(random coil),在對鋅、銅離子滴定時未有發現顯著的二級結構變化。 本研究中也利用各種不同之結合公式擬合(fitting)自身螢光之滴定結果來探討金屬離子與不同β-類澱粉蛋白之解離係數,同時也將利用ITC滴定之結果以與其比較。最後發現整體結合公式(total binding equation)最能充分描述我們在自身螢光滴定實驗下所獲得的結果,原生型β-類澱粉蛋白與銅、鋅離子之解離常數分別為3.86、4.26μM,與ITC之結果3.79、4.27μM幾乎完全相同。 由本研究之結果發現,H6之突變型滴定曲線與其他類型相比確有較大之差異存在,然而是否代表其鍵結機構不同於其他類型,仍有待進一步查證。我們認為,若能完成光誘導交聯法及二維異核化學位移相關光譜之結果必能更進一步確認實驗之結果。 The previous studies show that β-amyloid peptide(Aβ), the product from amyloid precursor protein (APP) cut by two secretase sequentially, is the major cause of Alzheimer’s disease (AD). Misfolding of this peptide can lead to the formation of neurotoxic plaque in in human brain, causing the neuron damaged. This hypothesis is called the amyloid cascade hypothesis. Accordoing to the different times of onset, AD can be categorized into two types: familial and sprodic forms. For the familial type, the early onset of AD is attributed to the gene mutation.Also, it has been reported that the aggregation mechanism of familial Alzheimer’s disease (FAD) is different from others. In addition, the accumulation of Zn2+, Cu2+, Fe3+, and Al3+ is observed when investigating AD patient’s brain tissue, implying that metal ion may be one of the factor that induces the aggregation of Aβ. In our study, we investigated the interaction between Zn2+ or Cu2+ and WT or mutants, and tried to understand the binding coordination between them. First, we expressed and purified 15N labeled Aβ40 WT and mutants for 1H-15N HSQC experiments. Assuming that the cutting efficiency of the TEV protease is 100%, the yield of 15N labeled Aβ is estimated to be around 0.4 mg/L. We concluded that, due to the poor cutting efficiency of our TEV protease, we were not able to successfully separate fusion protein from pure protein using HPLC. Further improvement of our experimental conditions and procedures is needed. Next, we tried to titrate the wild type and mutation form of synthetic Aβ with metal ions and examine the differences using spectrometry and ITC. When interacting with zinc ion,our intrinsic fluorescence and bis—ANS fluorescence results showed that the binding behaviors of H6A and H6R peptides were significantly different from others. Also, we found in our bis-ANS fluorescence results that the hydrophobic exposure area was found to decrease upon binding with copper ion, whereas opposite trend was detected in the case of zinc ion, evidently suggesting that the binding actions of Aβ with copper ion and zinc ion are different. In addition, our CD data indicated that Aβ monomer from NaOH preparation exist mainly in random coil structure and its secondary structure was not considerably influenced by the addition of metal ions. Furthermore, apart from fitting the intrinsic fluorescence data against different model equations to estimate the magnitudes of dissociation constant between Aβ and metal ions, we also compared these estimated values of dissociation constant with the ones obtained from ITC experiment. We found that the total binding equation can best fit our results. The dissociation constant of Aβ with copper and zinc ions were determined to be 3.86 and 4.26 μM, respectively, which are very close to the values, 3.79 and 4.27μM, obtained from ITC measurements. In summary, our study showed that, while H6 mutants have siginifcatly different titration behavior from others, further confirmation is still needed to verify whether the coordination is different or not. We think performing the photo-induced cross-linking of unmodified proteins along with heteronuclear single quantum coherence spectroscopy can certainly aid in better understanding the underlying binding mechanism(s). |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7067 |
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顯示於系所單位: | 化學工程學系 |
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