雷射捕捉誘發細胞色素Ｃ澱粉樣纖維生成過程之螢光分析

Abstract

我們研究了細胞色素c單體、區域交換二聚體和雙硫鍵結二聚體的雷射捕捉誘導澱粉樣蛋白纖維生成過程。我們使用1064奈米的連續雷射束作為捕捉光源，並將其聚焦於細胞色素c / 重水溶液中。加入少量硫黃素T於溶液中作為澱粉樣蛋白纖維的指示劑，並且使用405奈米雷射作為ThT的激發光，在結合澱粉樣蛋白纖維後，於485奈米放出螢光。在捕捉雷射照射之下，在焦點處生成一約4-5微米大小之聚集體，並且放出大量螢光，由此我們證實澱粉樣蛋白纖維之形成。三種細胞色素c溶液均經由相似的過程形成澱粉樣蛋白纖維。澱粉樣蛋白纖維生成過程可以分為A到D四個階段。第一階段A為雷射照射之初，為觀察到明顯的聚集體生成。在階段B中，我們觀察到直徑為2-3微米的淺灰色聚集體，此時螢光強度沒有增加。階段Ｃ，雷射持續照射下，聚集體的顏色變深且體積略為縮小，此時螢光強度開始增加。我們認為澱粉樣蛋白纖維之核化於此生成。最後階段D，隨著螢光的大幅度增長，聚集體擴大到雷射焦點之外。每個階段的時間取決於細胞色素c的四級結構以及雷射之功率和極化影響。隨著雷射光功率的增加，促進區域交換的二聚體的澱粉樣纖維生成，因區域交換的二聚體異常解離成未折疊之單體。對於雙硫鍵結二聚體，由於二聚體俱有強雙硫鍵，抑制澱粉樣蛋白形成。另外，405奈米雷射的長時間或高功率照射，可以加速雙硫鍵結二聚體的澱粉樣纖維生成。我們認為405奈米雷射導致二聚體發生光反應，造成雙硫鍵分裂，形成單體。由澱粉樣纖維之SEM圖像，我們觀察到澱粉樣纖維束於單體與區結構域交換之二聚體，而雙硫鍵結二聚體觀察到除了纖維束之外的纏結結構似毛線團。這種通過雷射捕捉的澱粉樣纖維生成將有助於闡明澱粉樣纖維形成的動力學和機制。

We have studied laser trapping-induced amyloid fibrils formation dynamics of cytochrome c (cyt c) monomer, domain-swapped dimer, and S-S bond dimer. A continuous-wave laser beam of 1064 nm was used as a trapping light source and tightly focused into their cyt c/D2O solutions. A small amount of thioflavin T (ThT), which emits fluorescence at 485 nm upon binding to amyloid fibrils, was also added into the solutions as an indicator of amyloid fibrils, and a 405 nm laser was used as an excitation laser for ThT. After the trapping laser irradiation, 4-5 μm-sized single aggregate was eventually formed at the laser focus with a drastic enhancement of the fluorescence intensity. Based on this result, we have confirmed amyloid fibril formation, and all of the cyt c solutions exhibited almost same behavior of amyloid fibrils formation. The formation behaviors for the aggregate consisting of amyloid fibrils can be classified into four stages, from A to D. The first stage A can be seen in the early stage of the trapping irradiation, when nothing was apparently observed. For the stage B, we observed a pale grey aggregate with the size of 2-3 μm in diameter with no increase in the fluorescence intensity. The continuous irradiation into the aggregate suddenly made the color and size dark and small, when the fluorescence intensity started increasing. For this stage, we consider that nucleation for amyloid fibril takes place. Last stage D, the aggregate was grown and expanded to the outside of the laser focus with drastic fluorescence enhancement. The period for each stage depends on change in the quaternary structure of cyt c, critically affected by laser power and polarization. As the laser power is increased, the amyloid fibrils formation for the domain-swapped dimer is accelerated due to its efficient dissociation into unfolded monomers. For S-S bond dimer, the amyloid formation is restricted due to the strong disulfide bond of the dimer. It was also found that the amyloid fibril formation for S-S bond dimer can be accelerated by the longer irradiation time or higher power of the 405 nm laser. We consider that the 405 nm-laser irradiation causes the S-S-bond cleavage of the dimer into the component monomers through the photoreduction of the dimer. For SEM images of amyloid fibrils, we observed the bundles of the amyloid fibrils for monomer and domain-swapped dimer, while the tangled structure like a woolen in addition to the bundles of the fibrils was observed for S-S-bond dimer. We believe that this amyloid formation by laser trapping will be useful for elucidating the dynamics and mechanism of amyloid formation.