Department of Biotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
FEBS J. 2010 May;277(9):2038-50. doi: 10.1111/j.1742-4658.2010.07619.x. Epub 2010 Mar 19.
The conformational conversion of prion protein (PrP) from a native conformation to the amyloid form is a hallmark of transmissible spongiform encephalopathies. Conversion is usually monitored by fluorescent dyes, which bind generic amyloids and are less suited for living cell imaging. We report a new method for the synthesis of membrane-permeable and membrane-impermeable biarsenical reagents, which are then used to monitor murine PrP (mPrP) misfolding. We introduced tetracysteine (TC) tags into three different positions of mPrP, which folded into a native-like structure. Whereas mPrPs with a TC tag inserted at the N-terminus or C-terminus supported fibril formation, insertion into the helix 2-helix 3 loop inhibited conversion. We devised a quantitative protease-free method to determine the fraction of converted PrP, based on the ability of the fluorescein arsenical helix binder reagent to differentiate between the monomeric and fibrilized form of TC-tagged PrP, and showed that TC-tagged mPrP could be detected on transfected cells, thereby expanding the potential use of this method for the detection and study of conformational diseases.
朊病毒蛋白（PrP）从天然构象到淀粉样构象的构象转换是传染性海绵状脑病的标志。转换通常通过荧光染料进行监测，这些染料结合通用淀粉样蛋白，不太适合活细胞成像。我们报告了一种合成膜通透性和非通透性双砷试剂的新方法，然后用这些试剂来监测鼠朊病毒蛋白（mPrP）的错误折叠。我们在 mPrP 的三个不同位置引入了四半胱氨酸（TC）标签，这些标签折叠成类似天然的结构。虽然在 N 端或 C 端插入 TC 标签的 mPrP 支持纤维形成，但插入螺旋 2-螺旋 3 环会抑制转换。我们设计了一种定量的无蛋白酶方法来确定转化的 PrP 分数，该方法基于荧光素砷螺旋结合试剂区分 TC 标记的 PrP 的单体和纤维化形式的能力，并表明可以在转染的细胞上检测到 TC 标记的 mPrP，从而扩大了该方法在检测和研究构象疾病中的潜在用途。
