Structure-based Directed Evolution of Fast-Maturing GFPs

描述（由申请人提供）：项目概述。我们建议在结构和机制知识的指导下，通过定向进化开发快速成熟的绿色荧光蛋白。基于GFP技术的一个经常被注意到的严重限制是荧光信号的时间分辨率差。迄今为止，相当缓慢的荧光采集速度（最多半小时，通常是一小时）阻碍了将gfp样蛋白作为基因可编码探针用于实时视觉检测转录活性。这项工作旨在将GFP的成熟速度加快几倍，以改善基于GFP的融合标签的可用集，用于广泛的细胞和生物技术应用。本实验旨在提高绿色荧光蛋白生色团生物合成的化学步骤。我们实验室的结果表明，该过程的总体速率可能取决于三级蛋白质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将成为诱变的主要目标。将采用基于化学和结构约束的限制性随机化策略，然后对整个基因进行优化。使用我们实验室开发的自动激光扫描系统将有助于选择所需的特征。第一次，颜色获取率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。与公共卫生有关。虽然绿色荧光蛋白很容易检测，并且可以在细胞中精确定位，但它在实时监测转录调控方面的应用仍然有限。显然，成熟速度更快的gfp在通过高度调控的转录因子直接检测启动子激活方面具有巨大的优势。这种工具将有助于提高我们对干细胞分化和有机体发育的理解，以及与各种遗传和发育障碍相关的生物过程。更一般地说，快速成熟的绿色荧光蛋白将有助于开发大量疾病状态的治疗方法，包括神经退行性疾病和癌症疾病。

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.