Molecular Fluorescent Toolkit

描述（由申请人提供）：来自维多利亚水母的绿色荧光蛋白（GFP）及其来自珊瑚虫珊瑚的荧光同系物已成为细胞和组织体内成像的宝贵工具。珊瑚虫类GFP样蛋白质有不同的颜色和特征，因此为分子标记和细胞内检测提供了强大的新探针模板。一些珊瑚虫GFP样蛋白已经被开发成生物技术工具。然而，它们的光化学和低聚物性质限制了它们作为分子探针的有用性。我们的生色团形成机制和颜色决定因素的分析表明，单体蛋白质具有新的光谱和光化学功能可以设计。在现有的和新的单体红移荧光蛋白和色蛋白的基础上，我们计划开发两种类型的蛋白质标签，补充现有的GFP工具。其中包括目标1：单体荧光计时器，其随时间改变荧光颜色，和目标2：光活化荧光蛋白，其最初是暗的，但在用紫光照射时变成荧光。我们还将开发分子进化技术，包括候选蛋白的合理，组合和随机诱变，然后进行广泛的流式细胞术和多孔板光谱仪筛选。我们将把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进化，并将其应用于下一轮诱变。在目标3中，荧光变体将在哺乳动物细胞中作为融合标签进行彻底表征，并且先进的探针将用于人多巴胺转运蛋白的运输和内吞研究。阐明其细胞表面表达是如何调节的，将增强对正常神经传递以及药物成瘾等脑部疾病的理解。拟议研究的预期最终结果是一组分子荧光探针，将与GFP工具一样通用。这些探针将扩展GFP技术，允许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，这将导致新的定量方法的发展和活细胞的动力学显微镜的应用。

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.