Structural and mechanistic studies of PAS sensing

摘要 为了使细胞能够适当地适应周围不断变化的环境条件，它们必须依赖于感官， 这些蛋白质既能检测这些变化，又能启动适当的反应。作为应对这一挑战的一种解决方案， 自然界已经进化出配体调节的蛋白质/蛋白质相互作用结构域的类别，利用这些来控制 多种蛋白质功能。了解这些域如何进行必要的感知， 信号事件使人们对生物学的基本方面有了深入的了解，使新工具的开发成为可能， 用于生物医学研究，并启发了新的疾病疗法。在这里，我们重点研究的信号 含有PAS（Period-ARNT-Singleminded）结构域的蛋白质所使用的机制，在数十种 数千种蛋白质，它们控制超过20种酶和非酶效应物的活性 域.这些过程受到不同家庭成员不同刺激的调节， 结合辅因子的占据或构型的变化来“切换”活性的开启和关闭。的一些方面 这些触发物在某些PAS结构域中，例如在称为LOV（光-氧- 电压）感光域，其中蓝光照明驱动特定的光化学形成 蛋白质/黄素加合物，其变构控制发色团周围的蛋白质构象。然而，在这方面， 关于这些信号传导过程的基本问题仍然没有答案，限制了我们对 如何感知环境变化以及如何人为控制这些变化。我们建议回答 这些限制通过追求三个目标：1）。确定几种活化相关的结构变化 细菌LOV和PAS蛋白的种类; 2）.检查LOV信号在一类新的 真菌RGS-LOV蛋白; 3）.检查PAS对营养素中丝氨酸/苏氨酸激酶活性的调节- 传感人PAS激酶。为了实现这些目标，我们将利用广泛的基础， 初步的结构和功能数据，将与生物物理和生物化学的组合扩展 问题研究这项研究的结果将包括有关基本监管过程的信息 利用这些蛋白质，使深入了解生物医学的基本方面沿着与潜力 治疗和生物技术应用。

ABSTRACT For cells to properly adapt to changing environmental conditions around them, they must rely on sensory proteins which both detect these changes and initiate proper responses. As one solution to this challenge, nature has evolved classes of ligand-regulated protein/protein interaction domains, harnessing these to control numerous types of protein function. Understanding how these domains undertake the requisite sensing and signaling events has given insight into fundamental aspects of biology, enabled the development of new tools for biomedical research, and inspired novel therapies to disease. Here we focus on examining the signaling mechanisms used by proteins containing PAS (Period-ARNT-Singleminded) domains, found in tens of thousands of proteins where they control the activity of over 20 enzymatic and non-enzymatic effector domains. These processes are regulated by different stimuli in different members of the family, harnessing changes in the occupancy or configuration of bound cofactors to “switch” activity on and off. Some aspects of these triggers are well understood in some PAS domains, such as in the subset known as LOV (Light-Oxygen- Voltage) photosensory domains, where blue light illumination drives the specific photochemical formation of protein/flavin adducts which allosterically controls protein conformation around the chromophore. However, fundamental questions regarding these signaling processes remain unanswered, limiting our understanding of how environmental changes are sensed and how these might be artificially controlled. We propose to answer these limitations by pursuing three aims: 1). Determine activation-associated structural changes of several classes of bacterial LOV and PAS proteins; 2). Examine the generality of LOV signaling within a novel class of fungal RGS-LOV proteins; 3). Examine PAS regulation of serine/threonine kinase activity in the nutrient- sensing human PAS kinase. To achieve these ends, we will take advantage of a broad foundation of preliminary structural and functional data that will be extended with a mix of biophysical and biochemical studies. Outcomes from this research will include information about fundamental regulatory processes employed by these proteins, giving insights into both basic aspects of biomedicine along with potential therapeutic and biotechnology applications.