Molecular Fluorescent Toolkit

描述(申请人提供)：维多利亚水母的绿色荧光蛋白(GFP)及其来自珊瑚珊瑚的荧光同系物已成为细胞和组织活体成像的宝贵工具。与GFP变异体不同的是，珊瑚虫类GFP蛋白具有不同的颜色和功能，因此为分子标记和细胞内检测的新探针提供了强大的模板。几种类似珊瑚虫绿色荧光蛋白的蛋白质已经被开发成生物技术工具。然而，它们的光化学和低聚特性限制了它们作为分子探针的用途。我们对生色团形成机制和颜色决定因素的分析表明，可以设计出具有新的光谱和光化学特征的单体蛋白质。在现有和新型单体红移荧光蛋白和染色蛋白的基础上，我们计划开发两种类型的蛋白质标记，以补充现有的GFP工具。其中包括目标1：随时间改变荧光颜色的单体荧光计时器，以及目标2：可光激活的荧光蛋白，它们最初是暗的，但在紫光照射下变得荧光。我们还将开发分子进化技术，包括对候选蛋白质进行理性、组合和随机突变，然后进行广泛的流式细胞术和多孔板光谱仪筛选。我们将把突变过程与光谱和光化学变化联系起来，以深入了解负责荧光性质的生色团结构的分子进化，并将这些应用于下一轮突变。在目标3中，荧光变异体将作为融合标签在哺乳动物细胞中得到彻底表征，先进的探针将被用于人类多巴胺转运体的运输和内吞研究。阐明其细胞表面表达是如何调控的，将有助于加深对正常神经传递以及药物成瘾等脑部疾病的了解。这项拟议研究的预期最终结果是一套与GFP工具一样通用的分子荧光探针。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、动态和相互作用。这反过来将导致新的定量方法的发展和活细胞动态显微镜的应用。

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.