Small Molecule Tools to Image Sophisticated Protein Function

描述(申请人提供)：该申请寻求开发和应用超越荧光蛋白质的新的小分子策略，以成像体外或细胞内的蛋白质功能和离散的RNA，并创造影响生物学和医学发现的工具。一种工具是基于两部分四半胱氨酸(C4)展示，其中双砷染料的线性C4结合部位被分裂为折叠蛋白质的两个近似区域或蛋白质伙伴关系的两个成员。在第一个资助期间，我们报告了双砷化合物Flash和ReAsH首选的线性四半胱氨酸(C4)序列可以在保持高亲和力和亮度的同时在蛋白质伙伴关系的两个成员或蛋白质的两个近似区域之间分裂。随后，我们探索了二分C4显示器的结构要求，并将其应用于生成可编码的、荧光的无蛋白激酶传感器和P53救援剂传感器的原型，以及一种利用电子显微镜选择性成像蛋白质-蛋白质复合体的策略。这一更新要求继续支持两部分四半胱氨酸显示器的开发和应用，以及新发现的基于亲荧光双硼酸的正交标记策略。我们力求实现三大目标。第一个(目标1)是对通过详细的动力学、热力学、结构和光物理实验获得的两部分C4显示的更深入的、定量的理解。我们相信，其中获得的信息将提供信息并提高我们导航和解释这一应用中剩余实验的能力，并极大地促进新的基于两部分的实验和传感器的设计。目标2中的实验延续了前一个资助期的两个项目，它们具有最大的潜在影响。在Aim 2.1中，我们继续开发基于两部分C4展示的可编码酪氨酸激酶传感器，并共同应用它们来探索Abl激酶如何协调细胞骨架重排，以响应生长因子和黏附信号。在目标2.2中，我们继续开发稳定致癌p53变异的分子的传感器，并应用它们来识别新的p53小分子伴侣。目标3中的实验探索了双硼酸作为活体细胞成像中双砷化合物的无毒、非氧化还原替代品的潜力。我们将评估一组基于花菁的双硼酸作为标记富含丝氨酸的蛋白质模体的更明亮、更通用的替代方案，应用经过验证的选择方法来识别最佳序列标签。然后，我们将在这些结果的基础上开发可编码的RNA标签，并合作应用它们来可视化第二组内含子的移动和丙型肝炎病毒RNA基因组的运输。 与公共卫生相关：这项提案要求继续支持开发和应用两部分四半胱氨酸显示器和一种新的正交小分子标记策略，以荧光成像活细胞中离散的蛋白质构象和蛋白质组件。详细的动力学、热力学和光物理实验将使该方法建立在坚实的生物物理基础上。精心挑选的生物合作将通过应用程序展示监测Abl激酶、拯救不稳定的p53和监测丙型肝炎病毒基因组的贩运的效用。

DESCRIPTION (provided by applicant): This application seeks to develop and apply new small molecule strategies that go beyond fluorescent proteins to image protein function and discrete RNAs in vitro or inside the cell and create tools that impact discovery in biology and medicine. One tool is based on bipartite tetracysteine (C4) display, in which the linear C4 binding site for a biarsenical dye is split between two approximated regions of a folded protein or two members of a protein partnership. During the first funding period we reported that the linear tetracysteine (C4) sequence preferred by biarsenicals FlAsH and ReAsH could be split between two members of a protein partnership or two approximated regions of a protein while maintaining high affinity and brightness. Subsequently we explored the structural requirements of bipartite C4 display, and applied it to generate prototypes for encodable, fluorescent protein-free kinase sensors and p53 rescue agent sensors, as well as a strategy for the selective imaging of protein-protein complexes by electron microscopy. This renewal requests support for the continued development and application of bipartite tetracysteine display as well as a newly discovered orthogonal labeling strategy based on pro-fluorescent bis-boronic acids. We seek to achieve three major goals. The first (Aim 1) is a deeper, quantitative understanding of bipartite C4 display obtained through detailed kinetic, thermodynamic, structural, and photophysical experiments. We believe that the information obtained therein will inform and improve our ability to navigate and interpret the remaining experiments in this application and greatly facilitate the design of new bipartite-based experiments and sensors. The experiments in Aim 2 continue two projects from the previous funding period that possess the greatest potential impact. In Aim 2.1 we continue to develop encodable tyrosine kinase sensors based on bipartite C4 display, and apply them in collaboration to explore how Abl kinases coordinate cytoskeletal rearrangements in response to growth factors and adhesive cues. In Aim 2.2 we continue to develop sensors for molecules that stabilize oncogenic p53 variants, and applying them to identify new p53 small molecule chaperones. The experiments in Aim 3 explore the potential of bis-boronic acids as non-toxic, non-redox alternatives to biarsenicals for live cell imaging. We will evaluate a set of cyanine-based bis-boronic acids as brighter, more versatile alternatives for labeling serine-rich protein motifs, applying validated selection methods to identify optimal sequence tags. We will then build on these results to develop encodable RNA tags, and apply them in collaboration to visualize the mobility of Group II introns and trafficking of the hepatitis C virus RNA genome. PUBLIC HEALTH RELEVANCE: This proposal requests continued support to develop and apply bipartite tetracysteine display, and a novel orthogonal small molecule labeling strategy, to fluorescently image discrete protein conformations and protein assemblies in live cells. Detailed kinetic, thermodynamic, and photo-physical experiments will place the methodology on firm biophysical footing. Carefully chosen biological collaborations will showcase utility through applications to monitor Abl kinase, rescue destabilized p53, and monitor trafficking of the hepatitis C viral genome.