Department of Molecular Genetics, Federal State Budgetary Scientific Institution "Institute of Experimental Medicine", 197376 Akademika Pavlova St. 12, St. Petersburg, Russia.
Department of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute named by B. P. Konstantinov of National Research Center "Kurchatov Institute", 188300 mkr. Orlova roshcha 1, Gatchina, Russia.
J Struct Biol. 2019 Jan 1;205(1):78-83. doi: 10.1016/j.jsb.2018.11.006. Epub 2018 Nov 17.
In a number of conformational diseases, intracellular accumulation of proteins bearing non-native conformations occurs. The search for compounds that are capable of hindering the formation and accumulation of toxic protein aggregates and fibrils is an urgent task. Present fluorescent methods of fibrils' detection prevent simple real-time observations. We suppose to use green fluorescent protein fused with target protein and fluorescence lifetime measurement technique for this purpose. The recombinant proteins analyzed were produced in E. coli. Mass spectrometry was used for the primary structure of the recombinant proteins and post-translational modifications identification. The fluorescence lifetime of the superfolder green fluorescent protein (SF) and the SF protein fused with islet amyloid polypeptide (SF-IAPP) were studied in polyacrylamide gel using Fluorescent-Lifetime Imaging Microscopy (FLIM). It was shown that the SF average fluorescence lifetime in gel slightly differs from that of the SF-IAPP monomer under these conditions. SF-IAPP does not lose the ability to form amyloid-like fibrils. Under the same conditions (in polyacrylamide gel), SF and SF-IAPP monomers have similar fluorescence time characteristics and the average fluorescence lifetime of SF-IAPP in fibrils significantly decreases. We propose the application of FLIM to the measurement of average fluorescence lifetimes of fusion proteins (amyloidogenic protein-SF) in the context of studies using cellular models of conformational diseases.
在许多构象疾病中，会发生带有非天然构象的蛋白质在细胞内积累。寻找能够阻止有毒蛋白质聚集体和纤维形成和积累的化合物是一项紧迫的任务。目前用于检测纤维的荧光方法阻止了简单的实时观察。为此，我们拟使用与靶蛋白融合的绿色荧光蛋白和荧光寿命测量技术。分析的重组蛋白在大肠杆菌中产生。质谱用于重组蛋白的一级结构和翻译后修饰的鉴定。使用荧光寿命成像显微镜（FLIM）在聚丙烯酰胺凝胶中研究了超折叠绿色荧光蛋白（SF）和与胰岛淀粉样多肽（SF-IAPP）融合的 SF 蛋白的荧光寿命。结果表明，在这些条件下，SF 在凝胶中的平均荧光寿命与 SF-IAPP 单体的平均荧光寿命略有不同。SF-IAPP 并未失去形成类似淀粉样纤维的能力。在相同条件下（在聚丙烯酰胺凝胶中），SF 和 SF-IAPP 单体具有相似的荧光时间特性，并且纤维中 SF-IAPP 的平均荧光寿命显著降低。我们建议将 FLIM 应用于构象疾病细胞模型研究中融合蛋白（淀粉样蛋白形成蛋白-SF）平均荧光寿命的测量。
