Abstract
利用固態核磁共振光譜,熱差掃描分析儀及螢光技術的測量,來研究心臟毒素與中性脂質間的疏水性作用模式,材料上選用來自臺灣眼鏡蛇蛇毒中含量最高的三號心臟毒素CTX A3來和中性脂質(DPPC)作用,希望能找到兩者間的結合模式. 從結果分析,在脂質為漣波相時,蛇毒分子能夠以整個分子穿插到膜中,並推測可以將脂質多層膜融出像盤狀的小結構,而這個結構會受到磁場的作用而整齊排列於特定方向,在溫度升高後,這些盤狀結構又會融合在一起.當脂質升溫到液晶相時,因為脂質疏水性區間長度的改變,所以造成結合模式的改變,根據結果我們提出在液晶相時兩者間有兩種作用模式:(i)在較低溫時,蛇毒可以維持穿插到膜中的形式,(ii)較高溫時,蛇毒則和脂質間以周圍結合的方式作用.兩者間的飽和比例約在蛇毒與脂質比為10:200. 作用時,蛇毒會對脂質頭部做進一步的擾動,讓整個頭部的運動增快,同時蛇毒上的正電荷在不同的結合模式下,也會對頭部有不同的電荷效應,因而對頭部的角度造成不同的影響.而在測量光譜時,脂質頭部對磁場的夾角可能成魔角(約54度)的角度,以致於等方向性的訊號只在頭部訊號上呈現. A3 蛇毒屬於P-type的蛋白質,所以在三個環狀組成的疏水性區域,推測就是和脂質的尾部疏水區相結合的區域.而在研究兩者間的作用時,疏水區間的長度是否能符合是一個重要的關鍵,脂質尾部長度的些微變化就可以整個影響到蛋白分子與脂質的作用模式.The Interaction of Taiwan cobra cardiotoxin(CTX A3), a basicpolypeptide consisting of three fingered loops and five strandB-sheet structure, withzwitterionicdipalmitoylphophatidylcholine(DPPC) has been studied by 31P and2H NMR to understand the binding modes of CTX in membranebilayers. The resultsin conjunction with DPH fluorescenceanisotropy and differential scanning calorimetry studies, showthat CTX may penetrate and lyse the bilayers into smallaggregates at a lipid/protein molar ratio of about 20 in theripple Pb'phase. Elevating the temperature to that of the liquidcrystalline La phaseleads to the fusion of the small aggregatesinto larger ones as evidences bythe change of the isotropicsignal into magnetically aligned 31P signal with a markedreduction in the chemical shift anisotropy. 2H NMR study ondeuteriumlabeled DPPC in the head group and fatty acid region asa function of tempera-ture and CTX concentration reveals amolecular model that CTX undergoes a redistribution betweenpenetrating and peripheral binding states depending onthetemperature studies. In addition, both the comformational anddynamic stateof phosphocholine headgroup of DPPC bilayers aresignificantly pertubed in the presence of CTX. Structualconsideraton of CTX molecule indicates that the penetrationbinging mode of CTX with DPPC bilayer may involve a novelmembranebinding motif identified recently in the three-fingeredloops of P-type CTX.CTX can only bind to DPPC membraneperipherally in the La phase due to the mismatch of theirhydrophobic length.