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

这一建议旨在开发更好、更快和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”。它们负责驱动或执行细胞内大多数复杂和基本的活动。它们组装、移动、合成、催化、清洁和保护细胞内外的几乎所有东西。执行这些特殊功能需要数千种不同的蛋白质，每种功能都是由蛋白质独特的3D结构和特征运动决定的。了解蛋白质的结构、功能和动力学是过去50年来研究的热点。这种兴趣不仅仅是由科学好奇心驱动的。事实上，了解蛋白质的结构和功能是了解疾病、开发新药、创造新的生物制品、防治害虫和提高作物生产力的关键。因此，结构生物学已经获得了十几个诺贝尔奖，并且花费了100亿美元来确定大约9万种不同蛋白质的结构也就不足为奇了。然而，蛋白质结构的测定仍然是困难的、昂贵的、耗时的，而且经常充满错误。在过去的20年里，我的研究重点是设计和实验测试新技术，以改善蛋白质结构表征。在这段时间里，我们已经提出了许多非常优雅和简单的方法，极大地帮助加速和简化了通过核磁共振（NMR）光谱测定蛋白质结构。这些新颖的核磁共振方法现已被世界各地成千上万的结构生物学家广泛使用。对于这个提案，我计划改进我们之前的工作，并开始将最后的拼图拼凑在一起。我相信这种“最后的推动”最终将使蛋白质结构生物学的许多方面变得更快、更容易。在接下来的5年里，我的实验室将致力于3个特定目标：1)创建稳健的基于核磁共振化学位移的方法，用于一致和快速的3D蛋白质结构测定；2)设计新的基于核磁共振的方法来综合测量蛋白质动力学和热力学；3)最有趣的是，实现了一种基于质谱的方法，用于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，根据我们的初步数据，每个目标似乎都是可以实现的。方法的细节和我们的初步结果都包含在提案中。我们将使用的所有方法都是独特的，原创的，并建立在我们通过2009-14年NSERC资助取得的一些重要突破的基础上。如果我们实现了前两个目标，我们预计蛋白质结构测定、动态评估和热力学评估将加快3-4倍，成本将降低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，我们就可以打开一个结构生物学的全新领域，有朝一日可能与x射线晶体学或核磁共振光谱学相媲美。该项目为期5年，将为约10-12名学员（暑期生、研究生、pdf）提供极好的跨学科培训机会。所有学员都将有机会在结构生物学的前沿领域工作，操作高端核磁共振仪器和质谱仪，在湿（化学/生物化学）实验室和“干”（计算机）实验室工作，并学习机器学习和蛋白质化学的最新技术。他们还将能够应用这些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。

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.