Structure-based Directed Evolution of Fast-Maturing GFPs

描述(申请人提供)：项目摘要。我们建议在结构和机制知识的指导下，通过定向进化来开发快速成熟的绿色荧光蛋白(GFP)。基于GFP的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。到目前为止，相当缓慢的荧光获取速度(最多半小时，通常是一小时)阻碍了GFP样蛋白作为可编码的基因探针用于实时可视检测转录活动。这项工作旨在将GFP的成熟率提高几倍，以改进现有的基于GFP的融合标签集，用于广泛的细胞和生物技术应用。拟议的实验旨在加强GFP生色团生物合成的化学步骤。我们实验室的结果表明，这一过程的总体速度可能取决于位于三级蛋白质结构中靠近生色团形成氨基酸的碱基的强度。因此，直接生色团环境中的特定残留物将成为诱变的主要目标。基于化学和结构约束，将采用受限随机化策略，随后将对整个基因进行优化。通过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殖民地达到成熟终点所需的时间。与公共卫生的相关性。虽然GFP很容易检测，并允许在细胞中进行准确的定位，但它在实时监测转录调控方面的应用仍然有限。显然，具有更快成熟率的GFP在立即检测高度调控的转录因子激活启动子方面具有巨大的优势。这样的工具将有助于提高我们对干细胞分化和生物发育的理解，这是与各种遗传和发育障碍相关的生物过程。更广泛地说，快速成熟的绿色荧光蛋白将有助于开发针对包括神经退行性疾病和癌症疾病在内的大量疾病的治疗方法。

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.