ENGINEERING A YEAST SENSOR OF LIGAND-BINDING

小分子配体在蛋白质调节、信号转导和细胞中的其他基本过程中发挥重要作用，并且是治疗人类疾病的药物方法的基础。 因此，对于识别小配体结合的简单有效的方法有很大的需求，并且将在许多领域中具有应用，从药物发现中的高通量筛选到蛋白质-配体相互作用的结构研究。该提案概述了产生酵母温度敏感酶的计划，其活性受配体结合的调节。 这将是一种含有催化结构域报告基因和插入的或拴系的配体结合结构域的杂合酶。 该设计基于这样的前提，即配体与结构域的结合可以使其稳定或诱导变构变化，如果催化结构域紧密偶联，则可以通过催化结构域检测到变构变化。 为了鉴定具有偶联的结合和催化结构域的蛋白质，将在酵母中筛选工程化构建体，以获得在配体存在下显示改变的报告活性的形式。 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。 作为这种筛选的可行性的初步测试，将温度敏感性二氢叶酸还原酶（DHFR）与FK 506结合蛋白FKBP 12偶联，并在存在和不存在FK 506的情况下在酵母细胞中进行筛选。 如果这种策略是成功的，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。 理想地，杂合蛋白将具有模块化设计，使得其它结合结构域可以被取代以识别其它配体。

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.