心臟毒蛋白是眼鏡蛇蛇毒中含量最多的蛋白成分。其三維結構是由四
個雙硫鍵組成的核心區域加上向外延伸的三個指環區形成的弧形扁盤狀

結構，其中三個指環區是由兩組共五股的反平行β-褶板所構成的連續性疏水區；並可依圓二色光譜的特徵分成第一類與第二類心臟毒蛋白。

我們發現心臟毒蛋白在台灣東西岸不同的眼鏡蛇中有不同的分佈模

式，其中以心臟毒蛋白A6 的差別最為明顯。在這篇論文中我們以這個屬

於第一類的新心臟毒蛋白為例，運用核磁共振及圓二色光譜技術結合剛解

出的X 射線繞射結構對第一類心臟毒蛋白的第一指環區的構形做分析，得

知在水溶液環境下，第一類心臟毒蛋白是順式構形而第二類為反式構形。

心臟毒蛋白和核苷三磷酸的結合可能是心臟毒蛋白在活體外抑制蛋白

激酶C 與鈉-鉀-腺嘌呤核苷三磷酸水解酶活性的原因。我們用恆溫滴定熱

卡計及核磁共振的技術得知腺嘌呤核苷三磷酸是結合在心臟毒蛋白的凸

側上的陰離子結合區，而非文獻上提到的凹側。更指出序列上第五個位

置胺基酸的帶電性在腺嘌呤核苷三磷酸與心臟毒蛋白結合模式中有很大

的影響。這顯示胞內的腺嘌呤核苷三磷酸會與膜雙層內膜的磷脂質競爭

同一個陰離子結合位，因而促進心臟毒蛋白進入細胞，這也就是腺嘌呤核

苷三磷酸與心臟毒蛋白結合可能具有的生物意義。

Cobra cardiotoxin (CTX) is the major component of cobra snake venom. The three-dimensional structure of a CTX molecule consists of a rigid core region stabilized by four disulfide bonds and three protruding finger loops—loop I, II and III,
formed by five anti-parallel β-sheets. It is a basic polypeptide with a slightly concave disc-like shape. CTXs were classified into two classes by their distinct CD spectra.

There are different distribution pattern in Eastern and Western Taiwan cobra –Naja atra, the most significant one is the amount of CTX A6, a recently identified component of Taiwan cobra venom. In this thesis, we take this new CTX as a model

to study the loop I conformation of class I cardiotoxin. We showed that all results indicated the existence of a cis-peptide bond between Pro8-Pro9 by NMR, CD and analysis of the newly solved X-ray structure of CTX A6. This solves the discrepancy between the conformation of loop I in the NMR and X-ray structures of Tγ, the only class I CTX with both NMR and X-ray structures. According to our results, we conclude that the conformation of loop I of class I CTX will be the cis-conformer

rather than the trans-conformer of class II CTX.

It has been suggested that the binding of nucleotide triphosphate to CTXs may account for its in vitro activities of blocking the enzymatic activity of phospholipids protein kinase and Na+ K+-ATPase. Herein, we demonstrate by using ITC, NMR

chemical shift perturbation analysis and 1D selective excitation NOESY spectroscopic studies at pH ~6.0 that dATP binds to the convex side of CTX A1 & A3, CTX homologues from Naja atra, in sharp contrast to the previous suggestion of the dATP binding to the concave side of CTX A2 at pH 3.0. The ITC studies on the binding of ATP to 9 CTX homologues, shows the important role of positively charged residues of the 5th position in modulating CTX-ATP interaction.. It suggests that intracellular ATP may compete with the phospholipids of the inner leaflet of the membrane bilayer for binding on CTXs to facilitate their internalization.

In this thesis, we characterized the loop I conformation of class I CTX, shown an anionic binding pocket on the convex side of CTX, and role of Gln5 (Lys5 or Arg5), the biological meanings of loop I CTX and binding between ATP and CTXs

also were proposed. These will help further studies on the structure-function relationship of CTX.

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