Molecular Fluorescent Toolkit

分子荧光工具包

• 批准号: 7031132
• 负责人: Vladislav Verkhusha
• 金额: $ 35.28万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031132
• 关键词: analog; bioengineering /biomedical engineering; cell line; chromophore; combinatorial chemistry; dopamine transporter; endocytosis; flow cytometry; fluorescence resonance energy transfer; fluorescent dye /probe; green fluorescent proteins; high throughput technology; monomer; peptide chemical synthesis; photoactivation; protein transport; technology /technique development

DESCRIPTION (provided by applicant): The green fluorescent protein (GFP) from jellyfish Aequorea victoria and its fluorescent homologues from Anthozoa corals have become invaluable tools for in vivo imaging of cells and tissues. Anthozoa GFP-like proteins are available in colors and with features unlike those of GFP variants and, thus, provide powerful templates for new probes for molecular labeling and intracellular detection. Several Anthozoa GFP-like proteins have been already developed into biotechnological tools. However, their photochemical and oligomeric properties limit their usefulness as molecular probes. Our analysis of chromophore formation mechanisms and color determinants suggest that monomeric proteins with novel spectral and photochemical features can be designed. On the basis of existing and novel monomeric red-shifted fluorescent proteins and chromoproteins, we plan to develop two types of protein labels, complementary to the existing GFP tools. These include Aim 1: monomeric fluorescent timers that change fluorescent color with time, and Aim 2: photoactivatable fluorescent proteins, which are originally dark but become fluorescent upon irradiation with violet light. We will also develop molecular evolution techniques consisting of rational, combinatory and random mutagenesis of candidate proteins followed by extensive flow cytometry and multiwell plate spectrometer screening. We will correlate the mutagenesis process with spectral and photochemical changes to gain insight into the molecular evolution of chromophore structures responsible for fluorescence properties and apply these to the next rounds of mutagenesis. In Aim 3, the fluorescent variants will be thoroughly characterized in mammalian cells as fusion tags, and the advanced probes will be utilized in trafficking and endocytosis studies of the human dopamine transporter. Elucidation of how its cell surface expression is regulated will enhance understanding of normal neurotransmission, as well as brain diseases like drug addiction. The anticipated end result of the proposed research is a set of molecular fluorescent probes that will be as versatile as GFP tools. These probes will expand the GFP-technology to allow simultaneous detection of lifetime, dynamics and interaction of several proteins in a single cell. This, in turn, will lead to development of new quantitative methods and applications of a kinetic microscopy of living cells.

描述（由申请人提供）：来自水母的绿色荧光蛋白（GFP），来自维多利亚州的水母Aequorea及其荧光同源物来自Anthozoa珊瑚的荧光同源物已成为细胞和组织体内成像的宝贵工具。 Anthozoa GFP样蛋白具有颜色，具有与GFP变体不同的特征，因此为分子标记和细胞内检测提供了强大的模板。几种anthozoa GFP样蛋白已经发展为生物技术工具。但是，它们的光化学和寡聚特性限制了它们作为分子探针的有用性。我们对发色团形成机制和颜色决定因素的分析表明，可以设计具有新型光谱和光化学特征的单体蛋白。根据现有和新颖的单体红移荧光蛋白和铬蛋白的基础，我们计划开发两种类型的蛋白质标签，这些标签是与现有的GFP工具互补的。其中包括目标1：随着时间的变化荧光颜色的单体荧光计时器，AIM 2：可光活化的荧光蛋白，最初是黑暗的，但在用紫罗兰光照射后会变成荧光。我们还将开发由候选蛋白的合理，组合和随机诱变组成的分子进化技术，然后进行广泛的流式细胞仪和多孔板光谱仪筛选。我们将将诱变过程与光谱和光化学变化相关联，以深入了解负责荧光特性的发色团结构的分子演化，并将其应用于下一轮诱变。在AIM 3中，荧光变体将在哺乳动物细胞中彻底表征为融合标签，并且晚期探针将用于人类多巴胺转运蛋白的运输和内吞作用研究。阐明其细胞表面表达的调节方式将增强对正常神经传递的理解，以及诸如药物成瘾之类的脑部疾病。拟议的研究的预期最终结果是一组分子荧光探针，它们将与GFP工具一样通用。这些探针将扩展GFP技术，以同时检测单个细胞中几种蛋白质的寿命，动力学和相互作用。反过来，这将导致生物细胞动力学显微镜的新定量方法的发展。