Structure-based Directed Evolution of Fast-Maturing GFPs

描述（由申请人提供）：项目摘要。我们建议开发快速成熟的GFP（绿色荧光蛋白）的定向进化，指导结构和机械知识。基于GFP的技术的一个经常注意到的严重限制是荧光信号的时间分辨率差。迄今为止，荧光采集的相当缓慢的速率（最多半小时，通常一小时）已经阻碍了GFP样蛋白作为用于转录活性的实时视觉检测的可遗传编码的探针的使用。这项工作旨在加速GFP的成熟速率几倍，以改善用于广泛的细胞和生物技术应用的基于GFP的融合标签的可用集合。所提出的实验旨在增强GFP发色团生物合成的化学步骤。我们实验室的结果表明，这一过程的总体速率可能取决于蛋白质三级结构中靠近发色团形成氨基酸的碱基的强度。因此，直接发色团环境中的特定残基组将是诱变的主要靶标。基于化学和结构约束，将采用限制性随机化策略，然后优化整个基因。选择所需的功能将通过使用我们实验室开发的自动激光扫描系统来帮助。这是第一次，颜色采集的速度将直接用于初级文库的选择，激光技术将使我们能够确定数十万到数百万个菌落达到成熟终点所需的时间。与公共卫生的相关性。虽然GFP易于检测并允许在细胞中精确定位，但其在真实的时间中监测转录调节的用途仍然有限。显然，具有更快成熟速率的GFP在通过高度调节的转录因子立即检测启动子激活方面将具有巨大优势。这样的工具将有助于提高我们对干细胞分化和生物体发育的理解，这些生物过程与各种遗传和发育障碍有关。更一般地说，快速成熟的GFP将有助于开发用于包括神经退行性疾病和癌症疾病在内的大量疾病状态的疗法。

DESCRIPTION (provided by applicant): Project Summary. We propose to develop fast-maturing GFPs (green fluorescent proteins) by directed evolution, as guided by structural and mechanistic knowledge. An often-noted severe limitation of GFP- based technology is the poor temporal resolution of the fluorescence signal. To date, the rather sluggish rate of fluorescence acquisition (half hour at best, typically one hour) has hampered the use of GFP-like proteins as genetically encodable probes for the real-time visual detection of transcriptional activity. This work is aimed at accelerating the maturation rate of GFP several-fold, to improve the available set of GFP- based fusion tags for a wide range of cellular and biotechnological applications. The proposed experiments are designed to enhance the chemical steps of GFP chromophore bio-synthesis. Results from our laboratory indicate that the overall rate of the process may depend on the strength of a base positioned close to the chromophore-forming amino acids in the tertiary protein structure. Therefore, specific sets of residues in the immediate chromophore environment will be the primary targets for mutagenesis. A restricted randomization strategy will be employed based on chemical and structural constraints, and will be followed by optimization of the whole gene. Selection for desired features will be aided by the use of an automated laser scanning system developed in our laboratories. For the first time, the rate of color acquisition will be used directly in the selection of primary libraries, and the laser technology will allow us to determine the time required to reach the end point of maturation for hundreds of thousands to millions of colonies. Relevance to public health. Though GFP is easy to detect and allows for exact localization in the cell, it is as yet of limited use in monitoring transcriptional regulation in real time. Clearly, GFPs with more rapid maturation rates would be of tremendous advantage in the immediate detection of promoter activation by highly regulated transcription factors. Such a tool would help improve our understanding of stem cell differentiation and organismal development, biological processes that are related to a broad variety of genetic and developmental disorders. In more general terms, a fast-maturing GFP would aid in the development of therapies for a very large number of disease states including neurodegenerative and cancerous disorders.