本論文主要目的係藉由定點突變的方式探討KChIP2.2 (Kv channel
interacting protein2.2)四個EF-hands 與鈣離子及鎂離子結合能力對其結構之影響，及對Kv channel 結合反應之參與機制。CD spectra 顯示相較於突變EF-hands 1、2、3 及4，之重組蛋白之二級結構與wild-type 有明顯差異，但wild-type 及所有mutated KChIP2.2 之二級結構均可因結合鈣離子及鎂離子而有明顯變化。螢光實驗結果證實EF3 及EF4 為具有高親和性鈣離子結合的位置，但四個EF-hands 突變後均不影響KChIP2.2 與鎂離子之結合能力。膠體層析法結果顯示情形EF1、EF2 及EF3 突變後可使KChIP2.2 分子間引起oligomerization，而鈣離子及鎂離子可使其程度增加。突變蛋白及wild-type KChIP2.2 與猪腦組織萃取膜及liposome 之結合能力無明顯差異，且不受鈣離子及鎂離子的調控。Pull-down assay 結果顯示突變蛋白與KChIP2.2 在沒有離子存在下皆會與Kv4.2 結合，雖然鈣離子及鎂離子均會增加KChIP2.2 與Kv4.2 的結合，但 KChIP2.2 突變EF-hands 之後與Kv4.2結合能力明顯下降。細胞分佈實驗結果顯示突變EF1 後 KChIP2.2 集中於細胞核內，而其他EF-hand 突變蛋白及wild-type KChIP2.2 則分佈於細胞質與細胞核，提高細胞內鈣離子濃度會使突變後EF1 分佈至細胞質但對其他蛋白之分佈則無明顯改變。綜合上述結果顯示，四個EF-hands 扮演穩定KChIP2.2 構形之角色以利與Kv4.2 之結合反應，但此功能角色與細胞膜之結合能力無直接相關性。

Mutagenesis studies on the four EF-hands of KChIP2.2 (Potassium channel-interacting protein 2.2) were carried out to explore the conformational transition upon the binding of Ca2+ and Mg2+ and the subsequent effect on the interaction between KChIP2.2 and Kv4.2. CD spectra indicated that Ca2+- and Mg2+-loaded wild-type and mutated KChIP2.2 altered the secondary structure contents. In contrast to other mutants, mutation on EF1 caused a notably change in the secondary structure of KChIP2.2. Fluorescence measurement revealed that EF-hands 3 and 4 were high affinity Ca2+-binding sites within KChIP2.2 molecule, but the binding of Mg2+ with KChIP2.2 was marginally affected by EF-hand mutations. The results of size-exclusion chromatography showed that mutations on EF-1, EF-2 and EF-3 induced the oligomerization of KChIP2.2 and the extent of oligomerization was enhanced by Ca2+ and Mg2+. No significant differences were noted when wild-type and mutated KChIP2.2 bound with porcine brain membrane and liposome either in the absence or presence of Ca2+- and Mg2+. Pull down assay showed that KChIP2.2 and EF-hand mutants could bind with Kv4.2 in the absence of Ca2+ and Mg2+, but the interaction was enhanced by Ca2+ and Mg2+. However, the binding capability of mutants for Kv4.2 was notably lower than that observed for wild-type KChIP2.2. It was found that, in sharp contrast to that EF1 mutant exclusively localized in the nucleus, the other EF-hand mutants and wild-type protein distributed within nucleus as well as cytoplasm. Elevating intracellular Ca2+ concentration caused the translocation of EF1 mutant to cytoplasm but no appreciable effect on other mutants and wild-type KChIP2.2. . Taken together, these results suggest that the integrity of the four EF-hands are involved in function to stabilize conformation for binding with Kv channel, but this conformational transition is not essential for the binding to cell membrane.

目錄
中文摘要 ----------------------------------- 1
英文摘要 ----------------------------------- 2
英文縮寫表 --------------------------------- 3
序言 --------------------------------------- 4
實驗材料 ----------------------------------- 8
實驗方法 ----------------------------------- 11
實驗結果 ----------------------------------- 28
討論 --------------------------------------- 36
表 ----------------------------------------- 40
圖 ----------------------------------------- 45
參考文獻 ----------------------------------- 62

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