ENGINEERING A YEAST SENSOR OF LIGAND-BINDING

小配体在细胞内的蛋白质调节、信号转导和其他基本过程中发挥着重要作用，是治疗人类疾病的药物方法的基础。因此，识别小配体结合的简单而有效的方法是非常需要的，并将在许多领域得到应用，从药物发现的高通量筛选到蛋白质-配体相互作用的结构研究。这项提案概述了产生一种酵母温度敏感酶的计划，该酶的活性受配体结合的调节。这将是一种含有催化域报告和插入或连接的配体结合域的杂交酶。该设计是基于这样的前提，即配体与结构域的结合可以稳定它或引起变构变化，如果催化结构域紧密耦合，则可以检测到这种变化。为了识别具有结合和催化结构域的蛋白质，将在酵母中筛选出在配体存在时显示报告活性改变的形式的工程构建物。通过使用一种温度敏感蛋白质作为报告，可以在酵母中检测到催化活性或稳定性的细微变化。作为这种筛选可行性的初步测试，温度敏感型二氢叶酸还原酶(DHFR)将与FK506结合蛋白FKBP12偶联，并在有无FK506的酵母细胞中进行筛选。如果这一策略成功，就可以很容易地观察到酵母细胞温度敏感度的变化。理想情况下，杂交蛋白将具有模块化设计，以便可以替换其他结合结构域来识别其他配体。

Small ligands play important roles in protein regulation, signal transduction, and other fundamental processes in the cell, and are the basis for pharmaceutical approaches to treatment of human disease. Simple and efficient methods for recognizing binding of small ligands, therefore, are in great demand, and would have applications in a number of areas, from high-throughput screening in drug discovery to structural studies of protein-ligand interactions. This proposal outlines plans for generating a yeast temperature-sensitive enzyme whose activity is regulated by ligand binding. This will be a hybrid enzyme containing a catalytic domain reporter and an inserted or tethered ligand-binding domain. The design is based on the premise that ligand binding to a domain may stabilize it or induce an allosteric change that may be detected by a catalytic domain if it is closely coupled. To identify proteins with coupled binding and catalytic domains, engineered constructs will be screened in yeast for forms that show altered reporter activity in the presence of ligand. Subtle changes in catalytic activity or stability can be detected in yeast by using a temperature-sensitive protein as a reporter. As an initial test of the feasibility of such a screen, temperature-sensitive dihydrofolate reductase (DHFR) will be coupled to the FK506 binding protein, FKBP12, and screened in yeast cells in the presence and absence of FK506. If this strategy is successful, ligand binding can be easily observed as a change in temperature sensitivity of the yeast cells. The hybrid protein will ideally have a modular design, such that other binding domains can be substituted to recognize other ligands.