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研究生(外文):Kuan-Ming Chen
論文名稱(外文):NMR Study of Cobra Cardiotoxin on Conformational Equilibrium and Its Interaction with Phosphate Derivatives
指導教授(外文):Wen-guey Wu
外文關鍵詞:NMRCobra Cardiotoxinconformational equilibriumphosphate derivatives
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心臟毒蛋白分子是個帶正電的生物巨分子,可以和帶負電荷的分子如磷酸、脂質、及醣胺素等有很強的交互作用,最近發現這些磷酸衍生物可以抑制由心臟毒蛋白分子在紅血球上引起的溶血現象,因此決定心臟毒蛋白與磷酸衍生物的結合態,可進一步了解心臟毒素的毒理作用機制。雖然心臟毒蛋白分子和去氧腺核三磷酸 (dATP) 在酸性溶液下的複合體結構已被解出,本研究發現在近生理條件下心臟毒蛋白分子和dATP的複合體結構與在酸性溶液下有明顯的差異。運用核磁共振及電腦模擬,發現在中性溶液下dATP結合在心臟毒蛋白分子的凸面而非先前運用NMR在酸性溶液下的凹面。心臟毒蛋白分子和dATP的結合模式在酸性與中性溶液下的確不一樣這個結論,藉由螢光實驗也得到證實。
另外,我們也發現心臟毒蛋白分子 (CTX A3)有兩種構形的平衡存在,在25℃時,其數量比約為14.3%。藉由改變溫度及加入帶負電的三磷酸來觀察此兩種構形的比例變化。結果顯示,當溫度由5℃升到45℃時,其比例由22%降到12.4%。而當在25℃時,逐漸加入2mM的三磷酸,其比例由14.3%升到了31.6%。利用熱力學分析,得到造成兩種構形的主要驅動力是來自於熵值的變化。而由兩種構形的化學位移差異,我們推論導致這兩種構形產生的機制可能是由於酪胺酸 (tyrosine) 苯環引起,但至今還沒有相對應到分子結構上發生變化之證據。

The major toxin (CTX A3) from Taiwan cobra (Naja atra) is a basic polypeptide capable of depolarizing cell membrane and causing lytic effect on many cells, including cardiomyocytes. It binds to several negatively charged ligands, such as phosphate derivatives, phospholipids and glycosaminoglycans. With unknown function, since phosphate derivatives compounds could inhibit the CTX-induced hemolysis, it is interesting to determine the binding mode to understand their structural and functional relationship. Although the complex structure of CTX A3 and dATP at acidic pH has been determined, it is not clear whether the interaction of CTX A3 and phosphate derivatives under physiological condition would be the same. Based on the NMR study and computer simulation, we found that the dATP bound to the convex side of CTX A3 at neutral pH, in sharp contrast to the concave side of CTX A2 as determined previously at low pH. The existence of two different binding modes of the dATP/CTX A3 complex at different pH was also proved by using fluorescence technique.
We also found that two forms of CTX coexisted as major (M) and minor (m) form as detected in the 2D 1H NMR spectra of CTX A3. At 25℃, the m/M ratio was about 14.3%. By changing temperature, the m/M ratio decreased from 22.0% at 5℃ to 12.4% at 45℃. With addition of 2 mM tripolyphosphate at 25℃, it increased from 14.3 to 31.6%. Thermodynamic analysis suggests that the main driving force is due to the change of entropy. In addition, the chemical shift difference between two conformations indicates the mechanism of conformational transition may be derived from the tyrosine ring but the evidence corresponding to the change of molecular structure is not found yet.

I. INTRODUCTION…………………………..…………1
1.1 Snake Venom………………………………………….1
1.2 Structure of Cardiotoxin………………………………3
1.3 Origin of the Study on CTX A3 and
Phosphate Derivatives………………………………...4
1.4 Introduction to Phosphate Derivatives………………..5
1.5 Origin of Study on the Conformational
Equilibrium of CTX A3……………………………....5
2.1 Phosphate Derivatives………………………………...7
2.2 Purification of Cardiotoxin……………………………7
2.3 Preparation of NMR Samples…………………………7
2.4 Protein Chemical Shift Variation Analysis……………8
2.5 Molecular Modeling of the Complex…………………8
2.6 Hemolysis Assay…………………………………...…9
2.7 Fluorescence Measurements…………………………..9
3.1 Hemolytic Result of CTX A3 After Treated
With Different Kind of Phosphate Derivatives….……11
3.2 NMR Chemical Shift Perturbation Caused by
Phosphate Derivatives………………………….……..11
3.3 Location of dATP Binding Site……………….………13
3.4 Conformation of dATP……………………….……….14
3.5 Experimental Evidence for the Presence of Two
Conformations in Equilibrium………………….…….14
3.6 Equilibrium Constant Between the Major and
Minor Conformation of CTX A3…………….……….15
3.7 Study the Major and Minor Conformations of
CTX A3 After Binding with Tripolyphosphate……....17
IV. CONCLUSION…………………...……………….....18
4.1 The Interaction of CTX A3 and Phosphate

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