Development of Improved Methods to Rapidly Characterize Protein Structure, Function and Dynamics

开发快速表征蛋白质结构、功能和动力学的改进方法

• 批准号: RGPIN-2014-05438
• 负责人: Wishart, David
• 金额: $ 4.95万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=632590
• 关键词: Development; Improved; Methods; Rapidly; Characterize

This proposal is aimed at developing better, faster and cheaper methods for characterizing the structure, function and dynamics of proteins. Proteins are often called the “engines of life”. They are responsible for powering or performing most of the complex and essential activities inside cells. They assemble, move, synthesize, catalyze, clean and protect just about everything inside and outside the cell. Thousands of different proteins are required to perform these specialized functions and each function is determined by that protein’s unique 3D structure and its characteristic motions. Understanding the structure, function and dynamics of proteins has been the subject of intense research for the past 50 years. This interest is not just driven by scientific curiosity. Indeed, understanding protein structure and function is key to understanding disease, developing new drugs, creating new bioproducts, combating pests and enhancing crop productivity. It is little wonder then that a dozen Nobel prizes have been awarded for structural biology and >$10 billion has been spent determining the structure of ~90,000 different proteins. However, protein structure determination continues to remain difficult, expensive, time-consuming and often fraught with errors. For the past 20 years my research has focused on both devising and experimentally testing novel techniques for improving protein structure characterization. Over that time, we have come up with a number of very elegant and simple methods that have greatly helped accelerate and simplify protein structure determination via Nuclear Magnetic Resonance (NMR) spectroscopy. These novel NMR methods are now widely used by 1000s of structural biologists around the world. For this proposal, I plan to improve upon our earlier work and to start placing the final pieces of the puzzle together. I believe this “final push” will ultimately make many aspects of protein structural biology significantly faster and easier. Over the next 5 years my lab will work on 3 specific objectives: 1) create robust NMR chemical shift-based methods for consistent and rapid 3D protein structure determination; 2) devise new NMR-based approaches to comprehensively measure protein dynamics and thermodynamics; and 3) most interestingly, implement a mass spectrometry-based method for determining the 3D structure of proteins and protein complexes. Each of the objectives has clear performance goals and, based on our preliminary data, each objective appears to be attainable. Details of the methods and of our preliminary results are contained within the proposal. All of the approaches we will use are unique, original and build on some important breakthroughs we achieved through our 2009-14 NSERC funding. If we achieve our first 2 goals we expect protein structure determination, dynamic assessments and thermodynamic evaluations could be sped up by 3-4X and costs reduced by 50-90%. These new methods could be particularly useful for structure-based drug or pesticide design. If we achieve our third goal we could open a whole new field of structural biology that might someday rival X-ray crystallography or NMR spectroscopy. Over its 5-year lifetime, this project will provide superb interdisciplinary training opportunities for ~10-12 trainees (summer students, grad students, PDFs). All trainees will have the chance to work in cutting edge areas of structural biology, to operate high-end NMR instruments and mass spectrometers, to work in both wet (chemistry/biochemistry) labs and “dry” (computer) labs and to learn the latest techniques in machine learning and protein chemistry. They will also be able to apply this newly acquired knowledge to solve important problems that could profoundly affect the future of structural biology.

该建议旨在开发更好，更快，更便宜的方法来表征蛋白质的结构，功能和动力学。蛋白质通常被称为“生命的引擎”。他们负责为细胞内部的大多数复杂和基本活动提供动力。他们组装，移动，合成，催化，清洁和保护细胞内部和外部的几乎所有东西。需要数千种不同的蛋白质才能执行这些专业功能，并且每个功能都取决于该蛋白质独特的3D结构及其特征运动。在过去的50年中，了解蛋白质的结构，功能和动力学一直是激烈研究的主题。这种兴趣不仅是由科学的好奇心驱动的。确实，了解蛋白质的结构和功能是理解疾病，开发新药，创造新的生物产品，抗击害虫并提高作物生产力的关键。那时，毫无疑问，已经为结构生物学授予了十二个诺贝尔的价格，并且已经花费了100亿美元来确定〜90,000种不同蛋白质的结构。但是，蛋白质结构的确定仍然保持困难，昂贵，耗时，并且经常被错误。在过去的20年中，我的研究集中在设计和实验测试，以改善蛋白质结构表征的新技术。在这段时间里，我们提出了许多非常优雅和简单的方法，这些方法具有很好的帮助加速并通过核磁共振（NMR）光谱来加速并简化蛋白质结构的测定。现在，这些新型的NMR方法已被世界各地的1000种结构生物学家广泛使用。对于此建议，我计划改善我们的早期工作，并开始将难题的最后一部分放在一起。我相信，这种“最终推动”最终将使蛋白质结构生物学的许多方面变得更加快捷，更容易。在接下来的5年中，我的实验室将针对3个特定目标：1）创建强大的基于NMR化学移位的方法，以确定一致和快速的3D蛋白结构测定； 2）设计新的基于NMR的方法来全面测量蛋白质动力学和热力学； 3）最有趣的是，实施一种基于质谱的方法来确定蛋白质和蛋白质复合物的3D结构。每个目标都有明确的绩效目标，并且根据我们的初步数据，每个目标似乎都是可以实现的。该方法和我们的初步结果的细节包含在提案中。我们将使用的所有方法都是独特的，原创的，并以我们在2009 - 14年度NSERC资金中实现的一些重要突破为基础。如果我们实现了前两个目标，我们期望蛋白质结构确定，动态评估和热力学评估可以加速3-4倍，成本降低了50-90％。这些新方法对于基于结构的药物或农药设计特别有用。如果我们实现了第三个目标，我们可以打开一个全新的结构生物学领域，该领域可能有一天有可能风险X射线晶体学或NMR光谱。在其5年的寿命中，该项目将为约10-12名学员（夏季学生，研究生，PDF）提供出色的跨学科培训机会。所有学员将有机会在结构生物学的最前沿领域，运营高端NMR仪器和质谱仪，从事湿（化学/生物化学）实验室和“干燥”（计算机）实验室的工作，并学习机器学习和蛋白质化学方面的最新技术。他们还将能够运用这种新获得的知识来解决可能深刻影响结构生物学未来的重要问题。