Surveying protein partnerships and assembly with bipartite tetracysteine display

描述(由申请人提供)：本提案描述了一种小分子荧光策略，用于选择性地标记活细胞中离散的蛋白质构象和组装，并由此成像。我们将这一策略称为两部分四半胱氨酸展示。已知，含有线性四半胱氨酸序列Cys-Cys-Pro-Gly-Cys-Cys的重组蛋白被细胞渗透性、亲荧光的双砷染料Flash和ReAsH选择性标记。在这里，我们探索(目标1.1)线性四半胱氨酸序列是否可以在体外和活细胞中保持高生物亲和力和亮度的同时，在蛋白质伙伴关系的两个成员之间或单个蛋白质的两个近似区域之间分裂。接下来(目标1.2)我们量化当蛋白质或复合体错误折叠或错误组装时闪光/ReAsH亲和力和亮度的损失。最后，(目标1.3)我们描述了一种新的生物素-闪光结合物，旨在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体或折叠良好的蛋白质，允许丰富噬菌体或哺乳动物文库中折叠良好的蛋白质或稳定的蛋白质复合体。在目标2中，我们在目标1的基础上开发了可编码的两部分四半胱氨酸显示传感器，用于(目标2.1)酪氨酸激酶活性和(目标2.2)动态[Ca~(2+)]变化，这些传感器应该比类似的FRET传感器更明亮，并配备了额外的时间控制优势。以后的实验将开发类似的传感器，用于其他激酶、组蛋白甲基化和磷酸化。最后，(目标2.3)我们描述了基于四半胱氨酸显示的两部分传感器来识别在活细胞中拯救治疗相关的p53突变的分子。最后，在目标3中，我们提出了两部分四半胱氨酸显示的三个应用，充分利用了这一技术的独特特性。目的3.1描述复杂编辑的FAI(CEF)，在CEF中，闪光或ReAsH在照射后选择性地失活的不是先前报道的单个蛋白质，而是不同的蛋白质-蛋白质复合体。目的3.2描述了一种被称为络合物编辑聚合(CEB)的相关技术，在CEB中，ReAsH选择性地在不同的蛋白质-蛋白质复合体周围聚合二氨基联苯胺。最后，在目标3.3中，我们设计了分子来成像和荧光区分细胞膜上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，两部分四半胱氨酸展示选择性地标记、成像和/或灭活活细胞中离散的蛋白质构象状态和组装。拟议的实验应用两部分四半胱氨酸显示器来开发更明亮的可编码传感器，用于蛋白质激酶和[Ca~(2+)]的动态变化，用于检测体内蛋白质与蛋白质的相互作用，并用于高通量筛选稳定特定蛋白质折叠的化合物。它还可能提供一种方法来研究与阿尔茨海默氏症、帕金森氏病和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.