MIRA: Uncover Design Rules for Interaction and Assembly of Nature’s Molecular Machines

项目概要/摘要： 前所未有的高分辨率生物积木结构的出现正在彻底改变生物学的发展。 我们可以合理化蛋白质机制的方式，但结构只提供了一个必要的，但不充分的，开始 揭示蛋白质的性质、活性和功能。在理解上存在着根本性的知识差距 将蛋白质结构和表面化学转化为蛋白质性质和功能。即使是一个财产， 由于蛋白质溶解度不能从已知的蛋白质结构用我们目前的方法预先预测， 工具和知识，正如观察到的那样，许多单突变引起了最小的结构性 同时显著改变稳定性、性质和聚集性。我们的目标是揭开 用于将蛋白质表面结构特性转化为蛋白质表面活性、相互作用和功能。汉 实验室正在努力实现这种转换，其中包括先进的光谱学方法，探测 局部蛋白质动力学、位点特异性水合性质和构象集合。这些测量 通过现有的最先进的工具，如电子顺磁共振（EPR）线型 分析，脉冲偶极EPR和固态核磁共振（NMR），以及新的方法 Han实验室开发的，如Overhauser动态核极化（ODNP）和其他DNP放大 NMR方法。这些方法的结合将使蛋白质表面水化、拓扑结构的检测成为可能。 和相互作用，在生理条件下的稀溶液状态下，并具有增强的灵敏度。Han实验室 将系统地应用和完善的工具和方法来研究蛋白质的稳定性，相互作用，相分离， 低聚到聚集。该五年目标侧重于以下几类精选蛋白质。我们选择 以球状蛋白LOV为模型揭示结构-功能关系， 工程化导致诸如增强的结合亲和力、变构性、荧光性质和受控的 构象可塑性我们选择tau蛋白来研究一种内在无序蛋白（IDP）， 单点突变和微妙的伴侣相互作用显着调节其稳定性和聚集倾向 in disease疾病context上下文.我们的目标将是揭示机制，例如通过水合扰动或构象 系综移位，通过突变和其他修饰调节聚集倾向。最后我们 目的是揭示结构和机制的基础，以及功能的后果，低聚， 两种跨膜蛋白PR和A2 A。这项提案的长期目标是破译设计规则 用于蛋白质的相互作用和活性表面，以便根据蛋白质表面结构和拓扑结构可以 预测结合位点在哪里，或者学习如何设计一个合理调节蛋白质-蛋白质组装的结合位点， 并选择或设计特定的聚集途径。

Project Summary/Abstract: The emergence of unprecedented high-resolution structures of biological building blocks is revolutionizing the way we can rationalize the protein machinery, yet the structure only offers a necessary, but insufficient, starting point to uncover protein property, activity and function. There is a fundamental knowledge gap in understanding the translation of protein structure and surface chemistry into protein property and function. Even a property as widely studied as protein solubility cannot be a priori predicted from a known protein structure with our current tools and knowledge, as underscored by the observation that many single mutations invoke minimal structural changes while dramatically changing the stability, property and aggregability. Our vision is to uncover the code for translating protein surface structural properties into protein surface activity, interaction and function. The Han lab is working on achieving such translation, among others, aided by advanced spectroscopy methods that probe local protein dynamics, site-specific hydration properties and conformational ensembles. These measurements are enabled by existing state-of-the-art tools, such as electron paramagnetic resonance (EPR) lineshape analysis, pulsed dipolar EPR and solid-state nuclear magnetic resonance (NMR), as well as novel approaches developed by the Han lab, such as Overhauser dynamic nuclear polarization (ODNP) and other DNP-amplified NMR methods. The combination of these methods will enable the detection of protein surface hydration, topology and interaction, in dilute solution state under physiological conditions and with enhanced sensitivity. The Han lab will systematically apply and refine the tools and methods to study protein stability, interaction, phase separation, oligomerization to aggregation. The five-year goals focus on the following select classes of proteins. We choose the globular protein LOV as a model to uncover the structure-function relationship to aid in rational protein engineering leading to properties such as enhanced binding affinity, allostery, fluorescent property and controlled conformational plasticity. We choose the protein tau to study an intrinsically disordered protein (IDP) for which single-point mutations and subtle partner interactions significantly tune its stability and aggregation propensity in disease contexts. Our goal will be to reveal the mechanisms, e.g. by hydration perturbation or conformational ensemble shifts, through which mutations and other modifications modulate aggregation propensity. Finally, we aim to uncover the structural and mechanistic basis, as well as functional consequences, of oligomerization of two trans-membrane proteins, PR and A2A. The long-term goal of this proposal is to decipher the design rules for interactions and active surfaces of proteins, so that from the protein surface structure and topology one can predict where the binding site is, or learn how to design one that rationally modulates protein-protein assembly, and select or design specific aggregation pathways.