Surveying protein partnerships and assembly with bipartite tetracysteine display

描述（由申请人提供）：该提案描述了一种小分子荧光策略，用于选择性标记活细胞中的离散蛋白质构象和组装，从而对其进行成像。我们将这种策略称为二分四半胱氨酸展示。已知含有线性四半胱氨酸序列Cys-Cys-Pro-Gly-Cys-Cys的重组蛋白被细胞可渗透的前荧光双砷染料FlAsH和ReAsH选择性标记。在这里，我们探讨（目的1.1）是否线性四半胱氨酸序列可以分裂的蛋白质伙伴关系的两个成员之间，或一个单一的蛋白质的两个近似区域之间，同时保持高的双砷亲和力和亮度，在体外和活细胞。接下来（目标1.2），我们定量当蛋白质或复合物错误折叠或错误组装时FlAsH/ReAsH亲和力和亮度的损失。最后，（目的1.3）我们描述了一种新的生物素-FlAsH缀合物，其设计用于从活细胞中不太稳定的变体的混合物中捕获稳定的蛋白质复合物或良好折叠的蛋白质，从而允许富集噬菌体或哺乳动物文库中的良好折叠的蛋白质或稳定的蛋白质复合物。在目标2中，我们建立在目标1的结果，开发可编码的二分四半胱氨酸显示为基础的传感器（目标2.1）酪氨酸激酶活性和（目标2.2）动态[Ca 2 +]的变化，应该比类似的FRET传感器更亮，并配备了额外的优势，时间控制。以后的实验将为其他激酶、组蛋白甲基化和磷酸化开发类似的传感器。最后，（目标2.3）我们描述了基于双链四半胱氨酸显示的传感器，以识别分子，拯救治疗相关的p53突变体活细胞。最后，在目标3中，我们提出了利用这种技术的独特功能的二分四半胱氨酸显示的三个应用。目的3.1描述了复合物编辑的REMAI（CEF），其中FlAsH或ReAsH在照射后，选择性地结合不是单一蛋白质，如先前报道的，而是不同的蛋白质-蛋白质复合物。目的3.2描述了一种称为复合物编辑聚合（CEB）的相关技术，其中ReAsH选择性地聚合围绕不同蛋白质-蛋白质复合物的二氨基联苯胺。最后，在目标3.3中，我们设计分子来成像和荧光区分细胞膜中的替代受体酪氨酸激酶（RTK）构象状态。本申请描述了一种小分子荧光方法二分四半胱氨酸显示，以选择性地标记、成像和/或标记活细胞中的离散蛋白质构象状态和组装体。拟议的实验应用二分四半胱氨酸显示开发更明亮的编码传感器的蛋白激酶和动态变化[Ca 2 +]，在体内的蛋白质-蛋白质相互作用的检测，并为高通量筛选的化合物，稳定特定的蛋白质折叠。它还可以提供一种方法来研究与阿尔茨海默病和帕金森病以及囊性纤维化相关的早期蛋白质错误折叠事件。

DESCRIPTION (provided by applicant): This proposal describes a small-molecule fluorescence strategy to selectively label and thereby image discrete protein conformations and assemblies in live cells. We refer to this strategy as bipartite tetracysteine display. It is known that recombinant proteins containing the linear tetracysteine sequence Cys-Cys-Pro-Gly-Cys-Cys are selectively labeled by cell-permeable, pro-fluorescent biarsenical dyes FlAsH and ReAsH. Here we explore (Aim 1.1) whether the linear tetracysteine sequence can be split between two members of a protein partnership, or between two approximated regions of a single protein, while maintaining high biarsenical affinity and brightness, in vitro and in live cells. Next (Aim 1.2) we quantify the loss in FlAsH/ReAsH affinity and brightness when the protein or complex is misfolded or misassembled. Finally, (Aim 1.3) we describe a novel biotin-FlAsH conjugate designed to capture stable protein complexes or well-folded proteins from mixtures of less stable variants in live cells, allowing for the enrichment of phage or mammalian libraries for well-folded proteins or stable protein complexes. In Aim 2 we build on the results of Aim 1 to develop encodable bipartite tetracysteine display-based sensors for (Aim 2.1) tyrosine kinase activity and (Aim 2.2) dynamic [Ca2+] changes that should be brighter than analogous FRET sensors and equipped with the added advantage of temporal control. Later experiments will develop analogous sensors for other kinases, histone methylation and phosphorylation. Finally, (Aim 2.3) we describe bipartite tetracysteine display-based sensors to identify molecules that rescue therapeutically relevant p53 mutants in live cells. Finally in Aim 3 we propose three applications of bipartite tetracysteine display that exploit the unique features of this technique. Aim 3.1 describes complex-edited FALI (CEF), in which FlAsH or ReAsH, upon irradiation, selectively inactivate not single proteins, as reported previously, but distinct protein-protein complexes. Aim 3.2 describes a related technique called complex-edited polymerization (CEB), in which ReAsH selectively polymerizes diaminobenzidine surrounding a distinct protein-protein complex. Finally in Aim 3.3 we design molecules to image and fluorescently differentiate alternative receptor tyrosine kinase (RTK) conformational states in the cell membrane. The application describes a small molecule fluorescence approach bipartite tetracysteine display to selectively label, image and/or inactivate discrete protein conformational states and assemblies in live cells. The proposed experiments apply bipartite tetracysteine display to develop brighter encodable sensors for protein kinases and dynamic changes in [Ca2+], for the detection of protein-protein interactions in vivo, and for the high-throughput screening of compounds that stabilize specific protein folds. It may also provide a means to study early protein misfolding events associated with Alzheimer's and Parkinson's disease and cystic fibrosis.