Zhang Shouxiang, Hong Yuning
Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia.
Methods Mol Biol. 2022;2378:3-18. doi: 10.1007/978-1-0716-1732-8_1.
When proteostasis is challenged and becomes unbalanced, unfolded proteins can accumulate in the cells. Protein unfolding causes conformational changes and subsequent differentials in side-chain solvent accessibility and reactivity. In particular, when protein unfolds, non-disulfide-bonded cysteines that are usually buried in the native state can become surface exposed and thus accessible. A series of fluorogenic dyes including tetraphenylethene maleimide (TPE-MI) and its analogs were developed to capture cysteine exposure in unfolded proteins as a measure of unfolded protein load and proteostasis capacity in cells. These dyes are inherently non-fluorescent but show fluorescence turn-on effect when conjugated to unfolded proteins via reacting with exposed cysteines on the protein. Reacting with small biothiols such as glutathione does not induce fluorescence of these dyes. Here we describe the routine workflow to characterize unfolded proteins in vitro or unfolded proteomes in cells by TPE-MIs.
当蛋白质稳态受到挑战并变得失衡时，未折叠的蛋白质会在细胞中积累。蛋白质的展开会导致构象变化以及随后侧链溶剂可及性和反应性的差异。特别是，当蛋白质展开时，通常埋藏在天然状态下的非二硫键连接的半胱氨酸会暴露于表面从而变得可及。开发了一系列包括四苯乙烯马来酰亚胺（TPE-MI）及其类似物在内的荧光染料，以捕捉未折叠蛋白质中半胱氨酸的暴露情况，作为衡量细胞中未折叠蛋白质负荷和蛋白质稳态能力的指标。这些染料本身不发荧光，但当通过与蛋白质上暴露的半胱氨酸反应与未折叠蛋白质结合时，会呈现荧光开启效应。与谷胱甘肽等小生物硫醇反应不会诱导这些染料产生荧光。在这里，我们描述了通过TPE-MI在体外表征未折叠蛋白质或细胞中未折叠蛋白质组的常规工作流程。
