| Summary: | 碩士 === 國立交通大學 === 應用化學系碩博士班 === 106 === 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.
|
|---|
