A protein design- and structure-guided interrogation of signal transduction mechanisms

项目总结/摘要 活细胞已经进化出复杂的机制来检测它们的环境和跨 生物膜，诱导生物行为的反应。尽管这些受体普遍存在， 我们对信号跨膜传播的离散机制的理解仍然是 进化在这一领域中，组氨酸激酶（HK）是细菌、真菌 以及调节生长、存活或致病性的植物。HKs感觉到不同的细胞外刺激， 信号穿过膜并通过多个子域，激活磷酸化级联反应， 诱导转录反应。早期的模型提出，HK是通过大的，刚性的身体移动后， 感知细胞外刺激随后的工作表明，信号在HK子域之间以 逐步的方式，往往通过蛋白质动力学的变化，通知假说，信号转导 是HK络合物亚结构域之间热力学耦合的结果。这进一步表明，许多 在HK信号传导的过程中可以采用构象。研究其分子和生物物理基础 HK特异性和信号转导，我们提出了一种结构和蛋白质设计方法来询问 能量阈值、传感器特异性和跨膜信号传导中的构象偏差。首先，理性和 从头设计将用于产生非原生的、可调谐的传感器域，以确定 什么样的配体诱导的能量反应足以启动信号传导。这将得到补充， 通过序列识别的合成正交传感器域的实验表征-以及 结构引导神经网络算法与此同时，我们将继续研究X射线晶体学和低温电子学， 显微镜来阐明HK复合物或分离的亚结构域在各种信号传导状态下的结构， 告知组装结构-构象-功能关系。这项研究将大大促进我们的 理解跨膜信号转导的能量学和动力学，同时提高我们的能力， 使用蛋白质设计和计算生物学来询问和设计复杂的生物物理机制。的 我提出的努力也将直接实现我博士后任期的培养目标，提供必要的技能 为我的独立研究生涯做准备，研究和工程信号转导机制。

Project Summary/Abstract Living cells have evolved intricate mechanisms to detect their environment and transduce signals across biological membranes, inducing responses in organismal behavior. Despite the prevalence of these receptors, our understanding of the discrete mechanisms by which signals are propagated across membranes is still evolving. In this area, histidine kinases (HKs) are a predominant class of membrane receptors in bacteria, fungi, and plants that regulate growth, survival, or pathogenicity. HKs sense diverse extracellular stimuli and transduce a signal across the membrane and through multiple subdomains, activating a phosphorylation cascade and inducing a transcriptional response. Early models proposed that HKs do so through large, rigid body shifts after sensing extracellular stimuli. Subsequent work indicates that signals are passed between HK subdomains in a step-wise manner, often through changes in protein dynamics, informing the hypothesis that signal transduction is the result of thermodynamic coupling between subdomains of the HK complex. This further implies that many conformations may be adopted in the course of HK signaling. To investigate the molecular and biophysical basis of HK specificity and signal transduction, we propose a structure and protein design approach to interrogate energetic thresholds, sensor specificity, and conformational bias in transmembrane signaling. First, rational and de novo design will be used to generate non-native, thermodynamically tunable sensor domains to determine what ligand-induced energetic response is sufficient to initiate signaling. This will be complemented with experimental characterization of synthetic, orthogonal sensor domains identified through a sequence- and structure-guided neural network algorithm. In parallel, we will pursue X-ray crystallography and cryo-electron microscopy to elucidate the structure of HK complexes or isolated subdomains in various signaling states, to inform assembly of structure-conformation-function relationships. This research will significantly advance our understanding of energetics and dynamics in transmembrane signal transduction while advancing our ability to use protein design and computational biology to interrogate and engineer complex biophysical mechanisms. The proposed efforts will also directly fulfill the training goals of my postdoctoral tenure, affording the necessary skills to prepare me for an independent research career studying and engineering signal transduction mechanisms.