臺灣博碩士論文加值系統

本研究中，我們研發了新的固相化學交聯方法學。在agarose beads 之上接上化學交聯試劑SAED，此反應劑可以選擇性地收集到生物分子聚體表面的蛋白質並進一步鑑定其身份。比較某一特定蛋白質在beads收集到的樣品與在原本總蛋白質中量的差異，可以得知該蛋白質在此生物分子聚體中空間分佈的資訊。本研究中我們先以一個已知結構的系統，synthetic three-layered protein complex，來驗證此新方法學的正確性。再利用此經驗證過的方法學，來分析豬大腦後突觸質密區組成蛋白質之間的排列結構。
我們決定了後突觸質密區之中5個主要蛋白質和19個次要蛋白質位於後突觸質密區的表面或內層。結果顯示，dynein intermediate chain、AMPA receptors、kainate receptors、N-cadherin、β-catenin、N-ethylmaleimide-sensitive factor、heat shock 70 kDa protein 8 isoform 2以及actin等蛋白質主要位居於表面。α,β-tubulin subunits、dynein heavy chain、MAP2A/2B、MAP2C/2D、spectrin、neurofilament heavy subunits、neurofilament medium subunits、heat shock 70 kDa protein 12A、α-internexin、dynamin-1 以及PSD-95位居於深度3.5 nm以內的區域。N-methyl-D-aspartate receptors 以及α-subunits of calcium, calmodulin-dependent protein kinase II位於表面但卻形成或位處於特殊的結構中。基於我們的實驗結果，並結合前人對於後突觸質密區組成蛋白質之間接合關係的了解，我們提出了PSD構造的分子模型。

Agarose beads carrying a cleavable, fluorescent, and photoreactive cross-linking reagent on the surface were synthesized and used to selectively pull out the proteins lining the surface of supramolecules. A quantitative comparison of the abundances of various proteins in the sample pulled out by the beads from supramolecules to their original abundances could provide the information on the spatial arrangement of these proteins in the supramolecule. The usefulness of these synthetic beads was successfully verified by trials using a synthetic protein complex consisting of three layers of different proteins on glass coverslip.
By using these beads, we determined the interior or superficial locations of 5 major and 19 minor constituent proteins in the postsynaptic density (PSD), a large protein complex and the landmark structure of asymmetric synapses in the mammalian CNS. The results indicate that α,β-tubulins, dynein heavy chain, microtubule-associated protein 2, spectrin, neurofilament H and M subunits, a hsp70 protein, α-internexin, dynamin and PSD-95 protein reside in the interior of the PSD. Dynein intermediate chain, AMPA receptors, kainate receptors, N-cadherin, β-catenin, N-ethylmaleimide-sensitive factor, a hsc70 protein and actin reside on the surface of the PSD. The results further suggest that the N-methyl-D-aspartate receptors and the α-subunits of calcium, calmodulin-dependent protein kinase II are likely to reside on the surface of the PSD with, however, unique local protein organizations. Based on our results and the known interactions between various PSD proteins from data mining, a model for the molecular organization of the PSD is proposed.

壹、緒論………………………………………………………………1
貳、材料與方法
一、藥品………………………………………………………………5
二、純化PSD和subcellular fraction ……………………………6
三、凝膠電泳分析 (SDS-PAGE) ……………………………………9
四、西方點漬法 (Western blot)…………………………………11
五、電子顯微鏡分析PSD結構………………………………………12
六、合成SAED-conjugated DADPA-agarose beads reagent……13
七、利用SAED-conjugated DADPA-agarose beads reagent收集PSD 表層蛋白 ……………………………………………………………14
八、二維蛋白質電泳 ………………………………………………15
九、MALDI-TOF MS 鑑定蛋白質身份………………………………16
十、合成three-layered protein complex (TPC)………………18
十一、利用SAED-conjugated DADPA beads收集TPC表面蛋白質.20
十二、使用原子力顯微鏡檢測TPC…………………………………20
參、結果
一、 SAED-conjugated DADPA beads 的合成……………………22
二、 證明SAED-conjugated DADPA beads 的可用性……………23
三、 PSD的純化、形態與成份鑑定 ………………………………25
四、 以 SAED 或 SAED-conjugated DADPA beads 標定PSD……27
五、 鑑定SAED-conjugated DADPA beads 收集到的PSD表面蛋白質 …………………………………………………………………29
六、 預估E (Exterior)/T (Total) ratios 的範圍……………30
七、 根據E/T ratios決定24種PSD蛋白質的表面或內層分佈 …32
肆、討論
一、 PSD的各種組成蛋白質的表面/內層分佈……………………35
二、 α-CaMKII在PSD中的分佈 ……………………………………36
三、 細胞膜蛋白質在PSD中的分佈 ………………………………37
四、 PSD的分子模型 ………………………………………………39
五、 本方法學未來展望……………………………………………42
伍、表
表一、MALDI-TOF MS 鑑定PSD蛋白質 ……………………………46
陸、圖
圖1 、 SAED-conjugated DADPA agarose beads 的合成與應用..48
圖2 、 Synthetic three-layered protein complex (TPC)的特徵鑑定，使用TPC來證明 SAED-conjugated DADPA beads的功能………50
圖3 、 豬大腦皮質純化所得PSD的形態與成份鑑定 ………………53
圖4 、 分別以 SAED 或 SAED-conjugated DADPA beads 標定PSD蛋白質…55
圖5 、 利用一維凝膠電泳和西方點漬法來分析PSD蛋白質及SAED-conjugated beads與PSD反應後收集到的蛋白質之組成……………58
圖6 、 利用二维凝膠電泳分析比較PSD蛋白質以及經由SAED-conjugated DADPA beads 收集的PSD表面蛋白質之間成份的差異..60
圖7 、 利用西方點漬法和二維蛋白質電泳分析, 共決定了24種PSD蛋白質的E/T ratios………………………………………………………62
圖8 、PSD的分子模型 …………………………………………………64
柒、參考文獻……………………………………………………………66

捌、附錄
附錄一、以固相化學交聯方法學研究紅血球細胞膜表面蛋白質……76
附錄二、利用不連續蔗糖梯度離心證明microtubule 和 actin filament與 PSD之間的交互作用………………………………………90

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