ENGINEERING A YEAST SENSOR OF LIGAND-BINDING

小配体在蛋白质调节、信号转导和细胞中的其他基本过程中发挥重要作用，是人类疾病药物治疗的基础。因此，识别小配体结合的简单而有效的方法是非常需要的，并且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。这一建议概述了计划产生酵母温度敏感酶，其活性是由配体结合调节。这将是一种杂化酶，它包含一个催化结构域报告和一个插入或栓连的配体结合结构域。该设计基于这样一个前提，即配体结合到一个结构域上可以稳定它或诱导一个变构变化，如果它是紧密耦合的，这个变构变化可以被催化结构域检测到。为了鉴定具有偶联结合和催化结构域的蛋白质，工程构建体将在酵母中筛选在配体存在下显示改变报告活性的形式。通过使用温度敏感蛋白作为报告基因，可以在酵母中检测到催化活性或稳定性的细微变化。作为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FK506存在和不存在的酵母细胞中进行筛选。如果这个策略是成功的，配体结合可以很容易地观察到酵母细胞的温度敏感性的变化。理想情况下，杂交蛋白将具有模块化设计，这样其他结合域可以被取代以识别其他配体。

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.