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標題: 鈣離子與旋光I蛋白對旋光C蛋白中央螺旋區與旋光T蛋白C-端分子區間的構型影響
Effect of Calcium and Troponin I on the Proximity Relationship between the Central Helix of Troponin C and C-terminal End of Troponin T
作者: 趙桓履
Chao, Huan-Lu
關鍵字: Troponin
出版社: 生命科學系
摘要: 最近的研究報告顯示 : 雞骨骼肌旋光蛋白分子構型有幾個重要的分子區域:旋光C蛋白-N端鈣離子調控區(sTnC1-97) ; 旋光C蛋白-中央螺旋結構(sTnC89-100) ; 旋光I蛋白-C端的抑制片段(sTnI104-115); 旋光I蛋白-C端的轉換片段(sTnI116-131); 以及旋光T蛋白(sTnT 200-245)與旋光I蛋白(sTnI58-102) 形成之IT arm。在無鈣離子鍵結,旋光C蛋白-中央螺旋結構沒有完整的分子構型;但當鈣離子與旋光C蛋白-N端結合時,旋光C蛋白-中央螺旋結構發生明顯的構型變化,使得原本鬆緩的分子結構延展支撐整個旋光蛋白。然而,心肌旋光蛋白的分子模型並沒有發現同樣的分子構型變化;這種鈣離子活化旋光蛋白—蛋白分子構型的變化似乎只存在骨骼肌。先前的研究報告中發現,鈣離子的分子調控區可能經由IT arm而傳遞鈣離子活化骨骼肌收縮之訊號。為了進一步證實此分子機轉,本研究論文利用大腸桿菌表現雞大胸肌細胞內的旋光C蛋白與旋光T蛋白;在旋光C蛋白-102 (中央螺旋結構區) 與旋光T蛋白-264(C 端) 各有一個cysteine可以標定硫氫鍵專一性的螢光物,並且利用螢光共振能量轉移(FRET)與Pyrene 螢光光譜兩種實驗方法測定蛋白—蛋白間相對位置的變化。實驗結果發現:鈣離子鍵結旋光蛋白增加旋光C蛋白-102與旋光T蛋白-264兩胺基酸間的相對距離約3~4 Ǻ,但在旋光C蛋白與旋光T蛋白所形成的雙複合體 ,兩胺基酸之間的距離並沒有明顯的變化。這樣的實驗結果可以推測鈣離子訊息的傳遞是在與旋光C蛋白結合後經由旋光I蛋白影響IT arm而再傳遞到旋光T蛋白C-端。
Chicken skeletal troponin (Tn) has been resolved into several domains: a regulatory head of TnC (sTnC 1-97); a central helix of TnC (sTnC 89-100); an inhibitory segment of TnI (sTnI 104-115); a switch segment of TnI (sTnI 116-131); and IT arm (sTnI 58-102 ; sTnT 200-245) of Tn complex. In the calcium-free state, the conformation of the central helix of TnC is disordered; while Ca2+ binding induces conformational changes and forms a long α-helix in the central helix. The extended conformation of the central helix separates the regulatory head and IT arm of Tn complex, and so to maintain the structure of whole troponin complex. Although the global organization of the subunits in skeletal Tn complex is similar to that in cardiac Tn complex, the distinct feature of the central helix of TnC only occurred in skeletal Tn complex but not in cardiac isoform. To further understand the molecular mechanism of Ca2+ induced conformational changes in Tn complex, we over-expressed chicken fast skeletal TnC and TnT in E. coli. TnC contains a cysteine residue at Cys-102, while TnT has a cysteine residue at Cys-264. Both residues were specifically labeled with a sulfhydryl-reactive fluorescence probe. Along with the techniques of pyrene excimer fluorescence and FRET, the relative position between TnC-Cys102 and TnT-Cys264 in the binary and ternary complex was measured in the presence and the absence of Ca2+. Data obtained indicated that the distance between TnC-Cys102 and TnT-Cys264 is Ca2+ increased about 3-4Å in the ternary complex but not significantly altered in the binary complex. These results might suggest that Ca2+ binding to TnC could convey to the C-terminal domain of TnT only through the interaction with TnI (IT arm).
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