Conformational Disorder in Protein Function and Pathogenesis

执行摘要：我的研究计划的首要目标是揭示蛋白质中驱动生物活性的分子机制--有时甚至是病理机制。我的团队的方法使用了内部开发的独特技术，使我们能够直接监测“活性”蛋白质，因为它们在分子水平上调节定义生命的过程。目前的建议建立在我们先前成功的基础上，使我们能够更深入地探索：(I)酶催化中的构象动力学；(Ii)内在无序蛋白质中的功能和病理；(Iii)蛋白质中活性调节的结构和动力学基础；(Iv)用于定量测量蛋白质相互作用的氢/氢交换。背景：经典的结构生物学工具(即X射线结晶学和经典结构核磁共振)提供了对蛋白质“天然结构”的精细详细的“快照”。然而，如果所有蛋白质实际上都像它们在这些“快照”中显示的那样是静态的，那么大多数蛋白质将完全不起作用。为了实现生物活性，蛋白质必须获得特定的、更高能量的构象，这些构象是通过被称为构象动力学的热驱动的波动来填充的类似天然结构的集合。我的团队开发了一套独特的基于质谱学的工具，提供了构象动力学的详细图像，以及几乎所有生物过程背后的构象变化。在此基础上，我们探索了驱动蛋白质中催化、络合、蛋白质折叠和活性调节的分子过程。影响：这项拟议的研究解决了一些基本问题，这些问题对我们在分子水平上理解蛋白质的功能和发病机制具有重要意义。这是雄心勃勃的，但直接建立在我在前两个发现阶段奠定的基础上，我的团队以传统的学术标准衡量产生了实质性的影响(在高影响力的期刊上发表了53篇文章，邀请了70多场演讲等)。我们的技术已被加拿大、美国、中国、以色列、新西兰、瑞典和英国的集团复制。我们还与众多行业合作伙伴合作，在将基本发现转化为实际应用方面取得了不同寻常的高度成功。正是由于这一成功，我们更广泛的研究计划现在包括了不同但严重相互交织的发现和知识转化/动员分支。这些分支机构表现出积极的反馈，使我们现在能够实施发现研究计划，在该计划中，我们对分子机制的基础探索对“现实世界”的应用、加拿大的商业轨迹以及最终极大地增强了HQP的培训和职业机会产生了切实的影响。

EXECUTIVE SUMMARY: The overarching objective of my research program is to uncover the molecular mechanisms that drive biological activity - and sometimes pathology - in proteins. My group's approach employs unique technologies developed in-house that allow us to monitor 'active' proteins directly as they mediate the processes that define life at the molecular level. The current proposal builds on our prior successes, enabling a deeper exploration of: (i) Conformational dynamics in enzyme catalysis  (ii) Function and pathology in intrinsically disordered proteins (iii) The structural and dynamic basis of activity modulation in proteins (iv) Hydrogen / deuterium exchange for quantitative measurements of protein interactions  BACKGROUND: The classical tools of structural biology (i.e., X-ray crystallography and classical structural NMR) provide exquisitely detailed 'snapshots' of the 'native structure' of proteins. However, if all proteins were actually as static as they appear in these 'snapshots', most would be completely non-functional. To achieve biological activity, proteins must access specific, higher energy conformations within an ensemble of 'native-like' structures that are populated via thermally-driven fluctuations known as 'conformational dynamics'. My group has developed a unique set of mass spectrometry-based tools that provide a detailed picture of conformational dynamics, and the conformational shifts that underlie virtually all biological processes. From this foundation, we explore the molecular processes that drive catalysis, complexation, protein folding and activity modulation in proteins. IMPACT: The proposed research addresses fundamental questions that are of great significance to our understanding of protein function and pathogenesis at the molecular level. It is ambitious, but builds directly upon the foundation laid out in my previous two discovery periods, where my group has had substantial impact as measured by conventional academic standards (53 publications in high impact journals, over 70 invited talks etc.). Our technologies have been replicated by groups in Canada, the US, China, Isreal, New Zealand, Sweden and the UK. We have also had an unusually high degree of success at translating fundamental discoveries to practical applications in collaboration with numerous industrial partners. It is because of this success that our broader research program now includes distinct but heavily intertwined branches for discovery and knowledge translation / mobilization. These branches exhibit positive feedback, allowing us now to implement a discovery research program where our fundamental explorations of molecular mechanisms have a tangible impact on 'real world' applications, Canadian business trajectories and, ultimately, greatly enhanced training and career opportunities for HQP.