細胞色素c雙硫鍵二聚體澱粉樣蛋白纖維形成的雷射捕陷現象

Laser trapping dynamics of amyloid fibril formation of cytochrome c disulfide dimers

羅珮華(Pei-Hua Lo)

指導教授 ： 杉山輝樹

國立交通大學/理學院/分子科學研究所/碩士(2018年)

摘要

我們呈現了以雷射捕陷誘導細胞色素c雙硫鍵二聚體的澱粉樣蛋白纖維形成。我們分別以E104C及A83C的雙硫鍵二聚體作為目標。以1064奈米波長之連續波雷射作為雷射捕陷的光源，並將其聚焦在細胞色素c/重水溶液中。樣品溶液中我們加入了硫磺素T作為檢測澱粉樣纖維蛋白的染劑，硫磺素T結合澱粉樣蛋白纖維後所發出的螢光波長約為490奈米，其中我們以405奈米波長雷射作為激發光。
E104C雙硫鍵二聚體的澱粉樣蛋白纖維形成的過程可以分為A到D四個階段。其中，A階段再細分為三個部分。在階段A1中，沒有觀察到任何東西。在階段A2中，有個直徑約4微米的聚集體在焦點處形成。在階段A3中，聚集體中心(直徑約為2微米)逐漸變深，並且發出微弱螢光。在階段B中，聚集體大小沒有改變但中心的穿透率變低，而此階段螢光強度開始上升。在階段C中，聚集體中心逐漸地縮小且顏色變得更深，此時螢光強度大幅地上升，此現象證實了澱粉樣蛋白纖維的形成。在階段D中，聚集體成長至直徑約十幾微米的大小，但此時螢光強度卻逐漸地下降。我們認為是聚集體的成長速度快於澱粉樣蛋白纖維的形成速度，因此澱粉樣蛋白纖維被聚集體給包覆。而我們以SEM和TEM確認纖維結構。
我們以A83C雙硫鍵二聚體進行相同的實驗。經由雷射照射後，與E104C雙硫鍵二聚體的現象雷同，先是在焦點處形成直徑約4微米的聚集體，接著聚集體中心變深。隨著持續的照射，聚集體在十幾秒內瞬間成長至直徑約9微米的大小。然而，在照射的過程中，並沒有觀察到螢光。此結果明確地表示A83C雙硫鍵二聚體並沒有形成澱粉樣蛋白纖維。根據這些結果，我們鑒於雙硫鍵所在位置是否能形成β摺疊來討論澱粉樣蛋白原纖維形成的動力學和機制。此研究結果所帶來的發現，將為闡明澱粉樣蛋白的形成提供新的見解。

關鍵字

雷射捕陷 ； 澱粉樣蛋白纖維 ； 細胞色素c ； 雙硫鍵二聚體

並列摘要

We here demonstrate laser trapping-induced amyloid fibril formation of cytochrome c disulfide dimers. The D2O solution of E104C disulfide dimer or A83C disulfide dimer was utilized as a target. A continuous-wave laser of 1064 nm was used as a trapping light source, and tightly focused into the sample solution. The thioflavin T (ThT), which shows the peak of the fluorescence emission at 490 nm upon binding to amyloid fibrils, was add into sample solutions as an amyloid fibril dye, and a 405 nm-laser was introduced into the solution along the same optical path as the trapping laser.
We confirmed that the amyloid fibrils were formed by applying the trapping laser into the sample solution of E104C disulfide dimer. The process of the amyloid fibril formation is divided into the following four stages from A to D. The stage A is subdivided into the following three parts. At stage A1, nothing is observed after laser irradiation. At stage A2, a single aggregate of native protein (ANP) of the dimer is formed at the laser focus, when the size was measured to be about 4 μm in diameter. At stage A3, the central part (about 2 μm) of the ANP gradually changes dark, when the weak fluorescence emission is observed. At stage B, the transmittance at the central part of the ANP becomes lower with the irradiation without change in the size, and the fluorescence intensity starts to increase slightly. At stage C, the central part starts to shrink and become darker, when the fluorescence intensity is tremendously enhanced, supporting that amyloid fibrils are formed. At stage D, the assembly (consisting of the ANP and amyloid fibrils) grows up to about ten micrometers in diameter, however the fluorescence intensity gradually decreases. We consider that the growth rate of the ANP is higher than that of the amyloid fibril formation, thus the amyloid fibrils may be encompassed by the dimer ANP on the surface of the assembly. The fibril structure was identified by SEM and TEM observation, by which we concluded that the amyloid fibrils of E104C disulfide dimer was produced by laser trapping.
The same experiment was carried out for A83C disulfide dimer. As same as the case of E104C disulfide dimer, laser irradiation firstly produced a 4 μm-sized single ANP of the dimer at the laser focus, and then the central part of the ANP became dark. Then, the continuous irradiation into the ANP led to the sudden growth to about 9 μm, however no fluorescence emission was observed during laser irradiation. This result clearly indicates that amyloid fibrils are not formed for A83C disulfide dimer. Based on these results, we discussed the dynamics and mechanism of amyloid fibril formation in view of whether the structural region depending on the position of disulfide bond can form β-sheet. These findings in this work will give new insights into the elucidation of the mechanism of the amyloid fibril formation.

並列關鍵字

Laser trapping ； amyloid fibril ； cytochrome c ； disulfide dimer

參考文獻

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