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標題: Pyrene 螢光光譜分析骨骼肌蛋白 TnI N-端立型結構變化:鈣離子與蛋白-蛋白間的影響
Study of Pyrene Spectrofluorometry with Conformational Changes in the N-terminal Domain of Chicken Skeletal Troponin I Associated with
作者: 陳名偉
Chen, Ming-Wei
關鍵字: pyrene;骨骼肌旋光蛋白;sTnI;Excimer fluorescence;立型結構
出版社: 生物化學研究所
本研究利用對硫氫鍵有專一性之 pyrene 螢光物來標定TnI N-端的兩個硫氫鍵胺基酸 (Cys-48、Cys-64),並藉此測試 TnI 與其他肌絲蛋白分子間之交互作用,以及鈣離子鍵結 TnC 對 TnI 構型之影響。單分子標計之 pyrene 螢光物被特定能量波長 (345 nm) 光源激發後,在波長區間 390~400 nm 釋放出特有雙峰波型之螢光稱為 Monomer fluorescence。當兩相鄰 pyrene 分子相距在 3~5 Å 距離時,可藉由互相作用之游動電子共振形成激發態雙合體 (Excited Dimer) 而在波長 480~490 nm 區間釋放出一寬波的螢光稱為 Excimer fluorescence。Excimer fluorescence 決定於兩相鄰 pyrene 分子間趨近程度,因此可用來研究兩個相鄰胺基酸在空間上立型結構的變化。本研究論文分析 pyrene 螢光標定之 TnI 蛋白分子之螢光光譜所得結果如下:1.) Excimer fluorescence 的產生表示TnI N-端之 Cys-48 和 Cys-64 可能非常靠近因而易於形成 Pyrene Excimer。2.) 標定 TnI 與 TnT 或 TnC 鍵結,造成 Excimer fluorescence 顯著改變,意指著蛋白—蛋白間交互作用可互相影響進而造成 TnI N-端分子立型結構變化。3.) 當螢光標定 TnI 與 TnT/TnC 形成穩定的複合物 (Complex),鈣離子進一步鍵結此複合體會造成 Excimer fluorescence 減少 (~10.7 %)、 Monomer fluorescence 增加 (~6.2 %);此結果表示: TnI N-端分子區域可能參與鈣離子活化肌絲蛋白分子間的收縮機制。

The aim of this study was to characterize conformational changes in the N-terminal domain of troponin I (TnI) and to relate such changes to its regulatory functions of skeletal muscle contraction. Two Cys residues (Cys-48 and Cys-64) in the N-terminal domain of chicken skeletal TnI allow to attach conformational probes to this region. By analyzing spectrum of fluorescence probes attached at these sites, conformational changes in TnI. were characterized in association with protein-protein interactions in the presence or absence of calcium binding. Compounds which contain the pyrene group, such as N-(1-pyrene)maleimide (PM), N-(1-pyrene)iodoacetamide (PIA), and 1-pyrenemethyl iodoacetamide (PMIA) were used to study proximity relations between the two SH groups. The pyrene-labeled protein displays a characteristic fluorescence spectrum: two sharp peaks in the range from 380 to 400 nm, referred to as monomer fluorescence and one broad peak in the range from 480 to 490 nm, referred to as excimer fluorescence. The excimer fluorescence arises from the tendency of two adjacent pyrene groups to form an excited state dimmer. Thus the magnitude of the excimer fluorescence indicates the ability of the two pyrene groups to approach to within about 3~5 of each other. Results obtained are the followings: 1.) Two Cys residues (Cys-48 and -64) are close to each other and form pyrene excimer fluorescence. 2.) Interactions of TnI with TnT or TnC cause significant changes in excimer fluorescence, suggesting protein-protein interactions would affect the proximity of the two labeled Cys of TnI. 3.) When labeled TnI forms complex with TnT, and TnC, calcium binding causes a further decrease in excimer fluorescence (~10.7 %) and a small increase in monomer fluorescence (~6.2 %). Thus, the N terminal region of TnI may participate in the calcium-regulated skeletal muscle contraction.
Appears in Collections:生物化學研究所

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