鞘糖脂在細胞膜上的量雖少但他卻扮演一些重要的功能如脂質膜運輸及細胞訊號傳遞等因此他是細胞不可缺乏的成分。糖脂頭部的糖化則是非常多樣化，無論是頭部的大小、成分及其脂肪酸的長度。擁有複雜且較大的頭部的GM1，他的頭部必定比磷脂的膽鹼還要突出因而有比較可能性跟蛋白質做接觸。然而對於有一些糖脂如半乳糖神經酸胺、葡萄糖神經酸胺、及硫肝脂，其頭部的大小則是小於或等於磷脂的膽鹼。因此蛋白質是如何由多數磷脂質形成的膜當中辨認出糖脂還待理清。本論文利用固態及液態核磁共振實驗來證明硫肝脂的頭部的確比磷脂的膽鹼突出。我們成功的辨認出硫肝脂的水脂介面上的氫與十二烷基磷脂微胞的膽鹼上的氫之間的分子間的NOE。此類的分子間NOE也可以在其他不同組合的微胞如SGC/LPC及GM1/DPC觀察到。雖然我們可以在微胞小球觀察到此現象但為胞的弧度比正常的細胞膜大，我們因此運用固態HRMAS實驗來觀察SGC/POPC在有或無膽固醇的多水多層膜，結果也顯示在50~600ms的時間內也有相同的分子間NOE。除此之外也依據由X-光繞射得到的SGC/POPC多層膜的膜厚度，我們推斷SGC有時會比磷脂還突出因而方便蛋白來辨認。
台灣眼鏡蛇中的主要心臟毒素CTX A3是beta摺板蛋白對心肌細胞膜的硫肝脂具有專一性，在脂質膜上打洞、造成膜外漏及內吞等作用。我們針對含有10%硫肝脂的磷脂質囊泡做螢光實驗，結果顯示硫肝脂會使CTX A3凝聚而形成30 Å 及10-2 秒的短暫的孔洞。H9C2細胞膜的膜片鉗電生理實驗不異而同的給相同的結果。接著我們進一步以核磁共振搭配上分子模擬方式研究CTXA3與含有硫肝脂微胞小球的週邊結合模式而進一步與X-光晶體在類式脂質膜環境下所解出的CTX A3□硫肝脂複合體做比較。核磁共振實驗結果顯示硫肝脂的頭部結構會受到CTX A3的結合而改變，其結合前後的結構會由彎曲(-sc/ap)變成伸展(sc/ap)結構。比較X-光繞射的雙合體與核磁共振的單體體結構，我們發現硫肝脂的總體方向必須有所改變才能夠由CTX A3單體進一步形成CTX A3雙合體。由於從頭到尾CTXA3摺疊方式並沒有大改變，我們提出下列的說法：埋在磷脂膜當中的硫肝脂可以改變其局部和全體的結構來達到CTX A3雙體化同時也改變了CTX A3與膜結合方位來促進CTX A3形成短暫孔洞及細胞內吞作用。
帶正電的三指環心臟毒素蛋白(CTX)已證實可以與各種不同的帶負電的小分子作用例如蛇毒中的檸檬酸鹽，糖胺素，硫化鞘醣脂，及核三磷酸。CTXA3與這些小分子作用後所引起的生化反應也大不相同包含毒素雙體化，保留在細胞表面，在膜上形成孔洞，細胞內吞以及阻礙激酶酵素反應。之前研究顯示同源性心臟毒素之間會針對不同負電小分子提供不同的結合位子也引起不同效應。在本論文我們利用核磁共振技術來研究無機磷酸根、三磷酸脫氧腺苷、由肝素中取得的四糖結合到Naja atra的CTXA1。CTX A1是新型心臟毒素會使骨骼肌的壞死。我們將在本論文裡證明CTX利用多變通的側鏈提共上述的小分子相同的結合位子。不同鏈長的肝素在與CTX A3單一結合區作用時也是運用這種多變通的結合模式。我們的結果顯示硫酸肝素與蛋白質的相互作用是靠整體的電荷聚集而並非靠他們的細部結構。在此我們也顯示說心臟毒素同源性蛋白的結構雖有四對雙硫鍵穩定住，靠近結合位子的非保守胺基酸卻可以針對小分子結合位子做細部微調來影響其結合及雙體化的能力。

Glycosphingolipids are minor but essential components of cell membrane that exhibit important biological function in membrane trafficking and cell signaling. These glycolipids however are highly diverse in their carbohydrate head group size, composition and their ceramide chain length. For glycolipids with big and complex head group such as GM1, their head group is far more extended out from choline head of PC, providing an efficient contact with proteins. However for some glycolipids such GalCer, GluCer, and sulfatide (SGC), their head group size is of similar size or even smaller than choline of PC (major component of cell membrane). How proteins can efficiently interact with these glycolipids remains to be illustrated. By using solution and solid state NMR, herein we provide evidence suggesting a protruding state of sulfatide head group. We identified the intermolecular NOEs between protons at interface region of SGC and proton of choline head of DPC micelles. Similar intermolecular NOEs also evidences for SGC/LPC and GM1/DPC micelle. To rule out possibilities that these glycolipids protruding out from PC micelle due to high membrane curvature of micelles, we further perform HRMAS experiments on fully hydrated SGC/POPC with or without cholesterol at multilamellar condition, showing that at mixing time of 50ms ~ 600ms, similar intermolecular NOEs can also be observed. Along with X-ray diffraction data that gives the membrane thickness information of SGC/POPC multilamellar, we conclude that SGC may dynamically protrude out from PC to provide an efficient mechanism for protein recognition.
The major cardiotoxin from Taiwan cobra (CTX A3) is a pore forming □-sheet polypeptide that requires sulfatide (sulfogalactosylceramide, SGC) on the plasma membrane of cardiomyocytes for the CTX-induced membrane leakage and cell internalization. Herein, we demonstrate by fluorescence spectroscopic studies that sulfatides induce CTX A3 oligomerization in sulfatide containing phosphatidylcholine (PC) vesicles to form transient pore with pore size and lifetime in the range of about 30 Å and 10-2 sec, respectively. These values are consistent with the CTX A3-induced conductance and mean lifetime determined previously by using patch-clamp electrophysiological experiment on the plasma membrane of H9C2 cells. We also derived the peripheral binding structural model of CTX A3□sulfatide complex in sulfatide containing PC micelles by NMR and molecular docking method and compare with other CTX A3□sulfatide complex structure determined previously by X-ray in membrane-like environment. The NMR results indicate that sulfatide head group conformation changes from a bent shovel (-sc/ap) to an extended (sc/ap) conformation upon initial binding of CTX A3. An additional global reorientation of sulfatide molecule is also needed for CTX A3 dimer formation as inferred by the difference between the X-ray and NMR complex structure. Since the overall folding of CTX A3 molecules remain the same, sulfatide in phospholipids bilayer is proposed to play an active role by involving its local and global conformational changes to promote both the oligomerization and reorientation of CTX A3 molecule for its transient pore formation and cell internalization.
Cobra cardiotoxins (CTXs) are three-fingered polypeptides with positively charged domains that have been shown to bind to anionic ligands of snake venom citrate, glycosaminoglycans, sulfoglycosphingolipid and nucleotide triphosphate with various biochemical effects including toxin dimerization, cell surface retention, membrane pore formation, cell internalization and blocking of enzymatic activities of kinase and ATPase. The reported anionic binding sites, however, are found to be different among different CTX homologues for potentially different CTX activities. Herein, by NMR studies of the binding of inorganic phosphate, dATP (stable form of ATP), and heparin-derived tetrasaccharide to Naja atra CTX A1, a novel CTX molecule exhibiting in vivo necrotic activity on skeletal muscle; we demonstrate that diverse ligands binding to CTXs could also occur at a single protein site with flexible side chain interactions. The flexibility of such an interaction is also illustrated by the available heparin□CTX A3 complex structures with different heparin chain lengths binding at the same site. Our results provide a likely structural explanation on how the interaction between heparan sufate and proteins depends more on the overall charge cluster organization rather than on their fine structures. We also suggest that the ligand binding site of CTX homologues can be fine-tuned by nonconserved residues near the binding pocket because of their flexible side chain interaction and dimerization ability, even for the rigid CTX molecules tightened by four disulfide bonds.

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Chapter 3: Cobra cardiotoxin/sulfatide binding modes on membrane surface suggest a role of glycosphingolipid conformational changes in its membrane pore forming activity
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Chapter 4: Structures of Heparin-Derived Tetrasaccharide Bound to Cobra Cardiotoxins: Heparin Binding at a Single Protein Site With Diverse Side Chain Interactions
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