Division of Molecular and Cellular Biosciences: Investigator-initiated research projects (MCB)

分子和细胞生物科学部：研究者发起的研究项目（MCB）

• 批准号: 1158347
• 负责人: Nikolai Skrynnikov
• 金额: $ 44.62万
• 依托单位: Purdue University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-15 至 2015-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1158347&HistoricalAwards=false
• 关键词: Division; Molecular; Cellular; Biosciences; Investigator

In the last decade it has become clear that dynamic disorder is prominently represented in the proteome. In particular, intrinsically disordered proteins (IDPs) are widely involved in signal transduction. In this role, they bind to their (structured) targets; in doing so they themselves acquire a measure of structural order. The details of the binding process are, therefore, of both fundamental and practical importance. The salient feature of the binding mechanism is that it often relies on electrostatic interactions. Initially, the IDP is pulled toward its target by long-range electrostatic forces, forming what is termed an electrostatic encounter complex. Starting from this point, it quickly finds the correct conformation and binds tightly to the target. To obtain insight into the structure/dynamics of the electrostatic encounter complex, the PI will study the binding of a proline-rich peptide (which serves as a minimal model for an IDP) to an adapter protein (the c-Crk N-SH3 domain). The original system is altered by introducing one or two point mutations into hydrophobic grooves of the adapter protein SH3 domain. This abolishes tight binding and shifts the equilibrium toward the intermediate state. The resulting increase in the population of the encounter complex makes it amenable to a nuclear magnetic resonance (NMR) study using chemical shifts, paramagnetic restraints, and relaxation data. This experimental dataset is used to construct an in silico model of the system. Toward this goal, a number of ~1 micro sec molecular dynamics (MD) trajectories of the complex are generated. These MD data are used as a source pool to form an "ensemble of MD trajectories", which is tailored to reproduce the experimentally measured NMR parameters. Of note, this model can rigorously predict the pieces of data that are inherently dynamic. Such experimentally calibrated MD ensembles are expected to provide a number of new insights into the structure and dynamics of disordered protein systems. Although IDPs constitute a broad and fundamentally important class of proteins, they receive little or no coverage in college textbooks. In this sense there is a perceptible and growing gap between the concepts emerging in the research laboratories and the classroom teaching. The PI will contribute to bridging this gap by (i) making presentations at several undergraduate institutions, namely Bradley, Carleton, Oberlin, Butler, and Juniata College, all of which are historically connected to Purdue; (ii) developing visual materials that can be used by other teachers; the main findings of this project will be presented in a form of MD-based movies, designed and rendered by undergraduate students recruited into the PI's laboratory; (iii) publishing article(s) in Scientific American and/or the Journal of Chemical Education to provide a broad audience with an accessible introduction to IDPs. When discussing IDPs with students, the PI will stress the element of "thinking outside the box" that led to the emergence of this new concept in protein science. The realization that active research constantly challenges the textbook dogmas should provide a source of inspiration for interested students and help to steer some of them toward a career in science. In particular, the PI will use this special opportunity to engage the students from underrepresented groups. In this effort he will rely on the continuing support from the local chapter of NOBCChE (National Organization for the Professional Advancement of Black Chemists and Chemical Engineers).

在过去的十年中，动态紊乱在蛋白质组中的显著表现已经变得很清楚。特别是，内在无序蛋白（IDPs）广泛参与信号转导。在这个角色中，它们绑定到它们的（结构化的）目标；在这样做的过程中，它们自己获得了一定程度的结构秩序。因此，装订过程的细节既具有基础意义，又具有实际意义。结合机制的显著特点是它往往依赖于静电相互作用。最初，IDP被远距离静电力拉向目标，形成所谓的静电相遇复合体。从这一点开始，它迅速找到正确的构象，并与目标紧密结合。为了深入了解静电相遇复合物的结构/动力学，PI将研究富含脯氨酸的肽（作为IDP的最小模型）与适配器蛋白（c-Crk N-SH3结构域）的结合。通过将一个或两个点突变引入适配器蛋白SH3结构域的疏水凹槽来改变原始系统。这就消除了紧密结合，使平衡态向中间态转移。由此产生的偶遇复合物数量的增加使其适合使用化学位移、顺磁约束和弛豫数据进行核磁共振（NMR）研究。该实验数据集用于构建系统的计算机模型。为了实现这一目标，生成了络合物的1微秒分子动力学（MD）轨迹。这些MD数据被用作形成“MD轨迹集合”的源池，该集合可用于重现实验测量的NMR参数。值得注意的是，该模型可以严格预测固有动态的数据块。这种实验校准的MD系统有望为无序蛋白质系统的结构和动力学提供许多新的见解。虽然IDPs构成了一个广泛而重要的蛋白质类别，但它们在大学教科书中很少或根本没有得到报道。从这个意义上说，在研究实验室和课堂教学中出现的概念之间存在着明显的、越来越大的差距。PI将通过以下方式弥合这一差距：(i)在几所本科院校进行演讲，即布拉德利、卡尔顿、奥伯林、巴特勒和朱尼亚塔学院，这些学院在历史上都与普渡大学有联系；（ii）制作可供其他教师使用的视觉材料；该项目的主要发现将以医学电影的形式呈现，由PI实验室招募的本科生设计和呈现；（iii）在《科学美国人》和/或《化学教育杂志》上发表文章，向广大读者介绍国内流离失所者。在与学生讨论国内流离失所者时，PI将强调“跳出框框思考”的元素，这导致了蛋白质科学中这一新概念的出现。认识到积极的研究不断挑战教科书教条，应该为感兴趣的学生提供灵感来源，并帮助引导他们中的一些人从事科学事业。特别是，PI将利用这一特殊机会与来自代表性不足群体的学生接触。在这项工作中，他将依靠NOBCChE（全国黑人化学家和化学工程师专业发展组织）当地分会的持续支持。