CryoEM studies of dynamic bacterial proteases

背景：蛋白质由化学构建块(氨基酸)的长链组成，折叠成精确的3-D结构，负责执行大多数细胞活动。不正确折叠或不需要的蛋白质必须被移除，这一活动在很大程度上是由分隔的蛋白酶完成的。这些桶状的酶与伴侣蛋白合作，这些伴侣蛋白展开客户蛋白，将它们传递给蛋白酶，被分解并回收为其氨基酸成分。一个在真核生物和原核生物中都存在的感兴趣的系统是蛋白酶体，它由一个被调节颗粒覆盖的蛋白酶核心颗粒(20S CP)组成。要了解原核蛋白酶体如何与这些调节颗粒协同工作，需要知道它的3D结构，因为它识别、展开和降解其客户蛋白质靶标。在过去的五年里，电子冷冻显微镜(CryoEM)已经成为获得蛋白质酶体等大型、动态、多亚单位复合体的高分辨率结构信息的一种可行的方法。通过改进样品制备方法和利用新的算法来分析动态复合体，甚至可以研究在其催化循环中填充的蛋白酶复合体的瞬时状态，以提供关于蛋白质降解的分子机制的信息。目的：该研究计划的长期目标是了解细菌区隔蛋白水解酶的结构和动力学如何使其发挥功能，并揭示与底物和调节分子的相互作用。具体的短期和中期目标集中在20S CP与分枝杆菌中发现的称为蛋白酶体相关ATPase(MPA)和细菌蛋白酶体激活剂(BPA)的调节颗粒的相互作用，以及ClpC1AAA解折叠酶与ClpP1P2蛋白酶的结合。意义：这些研究将提供对这些分子机器潜在生物物理特征的基本了解，为利用靶向蛋白水解酶可以提供的优势的未来生物技术应用铺平道路。这些技术将能够对蛋白质浓度提供精确的时间控制，后果从新的抗生素到影响细菌表型和行为的人工信号系统。参与这项研究计划的高素质人员(HQP)将学习蛋白质表达和纯化、电子显微镜操作以及低温电子显微镜结构确定和建模的尖端技术。HQP还将在补充生化和生物物理技术方面获得经验，这些技术是表征这些大分子机器所必需的。低温电子显微镜工具箱在结构生物学研究中特别受追捧，因为最近的进展为研究以前难以处理的蛋白质靶标打开了许多机会。

Background: Proteins are composed of long chains of chemical building blocks (amino acids) that fold up into precise 3-D structures and are responsible for carrying out the majority of cellular activities. Improperly folded or unwanted proteins must be removed, an activity carried out in large part by compartmentalized proteases. These barrel shaped enzymes co-operate with chaperones that unfold client proteins, passing them on to the protease to be broken down and recycled into their amino acid components. One such system of interest that is present in both eukaryotes and prokaryotes is the proteasome, which consists of a protease core particle (20S CP) capped by a regulatory particle. Understanding how the prokaryotic proteasome functions in concert with these regulatory particles requires knowing its 3D structure as it recognizes, unfolds and degrades its client protein targets. In the past five years electron cryomicroscopy (cryoEM) has emerged as a viable approach to obtaining high resolution structural information on large, dynamic, multi-subunit complexes such as the proteasome. By improving methods of sample preparation and utilizing new algorithms for analysis of dynamic complexes, even transient states of protease complexes that are populated during its catalytic cycle can be studied to provide information on the molecular mechanisms of protein degradation. Objectives: The long-term goal of this research program is to understand how the structure and dynamics of bacterial compartmentalized proteases enable their function, and to uncover interactions with substrates and regulatory molecules that modify their function. The specific short- and medium-term aims focus on interactions of the 20S CP with the regulatory particles found in mycobacteria called Proteasome-associated ATPase (MPA) and Bacterial proteasome activator (BPA), as well as the ClpC1 AAA+ unfoldase combined with the ClpP1P2 protease. Significance: These studies will provide a fundamental understanding of the underlying biophysical characteristics of these molecular machines, paving the way for future biotechnology applications that take advantage of the targeted proteolysis proteases can provide. Such technologies will be able to provide precise temporal control of protein concentrations, with consequences ranging from new antibiotics to artificial signalling systems effecting phenotypes and behaviour of bacteria. Highly qualified personnel (HQP) involved in this research program will learn skills in protein expression and purification, electron microscope operation, and cutting-edge techniques for cryoEM structure determination and modelling. HQP will also gain experience in complementary biochemical and biophysical techniques necessary to characterize these large macromolecular machines. The cryoEM toolset is particularly sought after in structural biology research, as recent advances have opened up many opportunities to study previously intractable protein targets.