Small Molecule Tools to Image Sophisticated Protein Function

对复杂蛋白质功能进行成像的小分子工具

• 批准号: 8115641
• 负责人: Alanna Schepartz
• 金额: $ 28.86万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8115641
• 关键词: Address; Adhesives; Affinity; Amino Acid Motifs; Binding; Binding Sites; Biological; Biology; Biotechnology; Boronic Acids; Cell Line; Cell surface; Cells; Cellular biology; Chemicals; Collaborations; Complex; Cues; DNA Sequence Rearrangement; Development; Drug Design; Dyes; Electron Microscopy; Fluorescence; Foundations; Funding; Genome; Goals; Growth Factor; Hand; Hepatitis C; Hepatitis C virus; Hydroxyl Radical; Image; In Vitro; Indoles; Introns; Kinetics; Knowledge; Label; Life; Medicine; Methodology; Methods; Microscopy; Molecular Chaperones; Monitor; Mutation; Oncogenic; Peptides; Phosphotransferases; Protein Conformation; Protein Engineering; Protein Tyrosine Kinase; Proteins; RNA; Reporting; Request for Proposals; Resolution; Serine; Side; Structure; Surface; System; Thermodynamics; Toxic effect; Variant; Viral Genome; Yeasts; base; cellular imaging; cyanine; design; foot; improved; member; molecular imaging; novel; protein aminoacid sequence; protein complex; protein folding; protein function; prototype; quantum; research study; response; sensor; small molecule; success; tool; trafficking; viral RNA

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.

描述（由申请人提供）：本应用程序旨在制定和应用新的小分子策略，这些策略超出荧光蛋白，以在体外或细胞内部成像蛋白质功能和离散的RNA，并创建影响生物学和医学中发现的工具。一种工具是基于双分四环素半胱氨酸（C4）显示的，其中，用于折叠蛋白的两个近似区域或蛋白质伙伴关系的两个成员之间的两种近似区域分配了Biarsenical染料的线性C4结合位点。在第一个资金期间，我们报告说，Biarsenicals Flash和Reash首选的线性四环素半胱氨酸（C4）序列可以分配在蛋白质伙伴关系的两个成员或两个蛋白质的两个近似区域之间，同时保持高亲和力和亮度。随后，我们探讨了两分C4显示屏的结构要求，并将其应用于为编码的，无荧光蛋白质的激酶传感器和p53救援剂传感器生成原型，以及通过电子显微镜对蛋白质 - 蛋白质复合物进行选择性成像的策略。该更新请求支持双方四环素状态显示器的持续开发和应用，以及基于利荧光型双孔酸的新发现的正交标记策略。我们试图实现三个主要目标。第一个（AIM 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对两分C4显示的更深入的定量理解。我们认为，其中获得的信息将为我们的浏览和解释本应用程序中的剩余实验的能力提供信息，并极大地促进了新的基于两部分的实验和传感器的设计。 AIM 2中的实验继续进行了两个项目，从上一个融资期开始具有最大的潜在影响。在AIM 2.1中，我们继续基于两分C4显示器开发可编码的酪氨酸激酶传感器，并将它们合作应用它们以探索ABL激酶如何响应生长因子和粘合性线索来协调细胞骨架重排。在AIM 2.2中，我们继续为稳定致癌p53变体的分子开发传感器，并应用它们以鉴定新的p53小分子伴侣。 AIM 3中的实验探索了双孔酸作为活细胞成像的Biarsenicals的无毒的非雷克斯替代品的潜力。我们将评估一组基于氰基的双孔酸作为标记富含丝氨酸蛋白蛋白基序的更明亮，更通用的替代方法，并采用经过验证的选择方法来识别最佳序列标签。然后，我们将建立这些结果以开发可编码的RNA标签，并将它们合作应用它们以可视化II组内含子的迁移率和丙型肝炎病毒RNA基因组的运输。 公共卫生相关性：该提案要求继续支持以开发和应用两部分四环素状态显示器以及一种新型的正交小分子标记策略，以在活细胞中荧光图像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把方法放在牢固的生物物理基础上。精心选择的生物合作将通过应用来展示实用程序，以监测ABL激酶，挽救稳定的p53并监测丙型肝炎病毒基因组的贩运。