CryoEM studies of dynamic bacterial proteases

背景：蛋白质由长链的化学构建块（氨基酸）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去除，这一活动在很大程度上是由区隔化的蛋白酶进行的。这些桶状的酶与伴侣蛋白合作展开客户蛋白，将其传递给蛋白酶分解并再循环成氨基酸成分。在真核生物和原核生物中都存在的一个这样的系统是蛋白酶体，它由蛋白酶核心颗粒（20S CP）组成，上面有一个调节颗粒。了解原核蛋白酶体如何与这些调节颗粒协同工作，需要了解其3D结构，因为它识别，展开和降解其客户蛋白目标。在过去的五年中，电子低温显微镜（cryoEM）已经成为一种获取大型、动态、多亚基复合物（如蛋白酶体）的高分辨率结构信息的可行方法。通过改进样品制备方法和利用新的算法来分析动态复合物，甚至可以研究蛋白酶复合物在其催化循环中填充的瞬态，以提供蛋白质降解的分子机制信息。目的：本研究计划的长期目标是了解细菌区隔化蛋白酶的结构和动力学如何使其发挥作用，并揭示与底物和调节分子的相互作用，从而改变其功能。具体的短期和中期目标集中在20S CP与分枝杆菌中发现的调节颗粒蛋白酶体相关atp酶（MPA）和细菌蛋白酶体激活剂（BPA）的相互作用，以及ClpC1 AAA+展开酶与ClpP1P2蛋白酶的相互作用。意义：这些研究将提供对这些分子机器潜在生物物理特性的基本理解，为利用蛋白酶所能提供的靶向蛋白水解的未来生物技术应用铺平道路。这些技术将能够提供对蛋白质浓度的精确时间控制，其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。参与本研究计划的高素质人才（HQP）将学习蛋白质表达和纯化，电子显微镜操作以及冷冻电镜结构测定和建模的尖端技术。HQP还将获得补充生化和生物物理技术的经验，这些技术是表征这些大型大分子机器所必需的。冷冻电镜工具集在结构生物学研究中特别受欢迎，因为最近的进展为研究以前棘手的蛋白质靶点开辟了许多机会。

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.