Department of Combinatorial Chemistry, Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine, 150 Zabolotnogo St., 03143, Kyiv, Ukraine.
J Fluoresc. 2010 Nov;20(6):1267-74. doi: 10.1007/s10895-010-0678-1. Epub 2010 May 19.
A key feature of Parkinson's disease is the formation and accumulation of amyloid fibrils of the natively unfolded protein α-synuclein (ASN) inside neurons. Recently we have proposed novel sensitive monomethinecyanine dye T-284 as fluorescent probe for quantitative detection of ASN amyloid fibrils. In this study the T-284 dye complex with ASN fibril was characterized by means of fluorescence anisotropy, atomic force microscopy and time-resolved fluorescence techniques to give further insights into the mode of dye interaction with amyloid fibrils. The fluorescence anisotropy of T-284 was shown to noticeably increase upon addition of aggregated proteins indicating on stable dye/amyloid fibril complex formation. AFM imaging of fibrillar wild-type ASN revealed differences in heights between ASN fibrils alone and in presence of the T-284 dye (6.37 ± 1.0 nm and 8.0 ± 1.1 nm respectively), that is believed to be caused by embedding of T-284 dye molecules in the "binding channel" running along the fibril. Fluorescence decay analysis of the T-284 in complexes with fibrillar ASN variants revealed the fluorescence lifetime values for T-284/fibril complexes to be an order of magnitude higher as compared to the free dye. Also, the fluorescence decay of free T-284 was bi-exponential, while dye bound to protein yields tri-exponential decay. We suppose that in complexes with fibrillar ASN variants T-284 dye might exist in different "populations" due to interaction with fibrils in different conformers and ways. The exact binding mode of T-284 with ASN fibrils needs further studies. Studied parameters of dye/amyloid fibril complexes are important for the characterization and screening of newly-developed amyloid-sensitive dyes.
帕金森病的一个主要特征是神经元内未折叠的α-突触核蛋白（ASN）的淀粉样纤维的形成和积累。最近，我们提出了新型敏感的单甲川菁染料 T-284 作为定量检测 ASN 淀粉样纤维的荧光探针。在这项研究中，通过荧光各向异性、原子力显微镜和时间分辨荧光技术对 T-284 染料与 ASN 纤维的复合物进行了表征，以进一步深入了解染料与淀粉样纤维相互作用的模式。T-284 的荧光各向异性在加入聚集蛋白后明显增加，表明稳定的染料/淀粉样纤维复合物的形成。对纤维状野生型 ASN 的 AFM 成像显示，ASN 纤维单独存在和存在 T-284 染料时的纤维高度存在差异（分别为 6.37±1.0nm 和 8.0±1.1nm），这被认为是 T-284 染料分子嵌入沿纤维运行的“结合通道”造成的。T-284 与纤维状 ASN 变体复合物的荧光衰减分析表明，T-284/纤维复合物的荧光寿命值比游离染料高一个数量级。此外，游离 T-284 的荧光衰减是双指数的，而与蛋白质结合的染料则产生三指数衰减。我们假设，在与纤维状 ASN 变体的复合物中，由于与不同构象和方式的纤维相互作用，T-284 染料可能存在于不同的“群体”中。T-284 与 ASN 纤维的确切结合模式需要进一步研究。研究染料/淀粉样纤维复合物的参数对于新开发的淀粉样敏感染料的特性和筛选非常重要。
