Conformational Disorder in Protein Function and Pathogenic Aggregation

结构生物学的工具(即X射线结晶学和结构核磁共振)为蛋白质的“天然结构”提供了极其详细的“快照”。然而，如果所有的蛋白质都像它们在这些“快照”中显示的那样是静止的，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。我的团队的研究目标是具体了解构象动力学是如何驱动蛋白质功能的，以及在某些情况下，如何采用致病结构，将其聚集在一起形成危险的聚集体，称为淀粉样蛋白。要做到这一点，我们需要知道这些更高的能量结构是什么样子的，但识别它们并不是一件容易的任务，因为它们往往是短暂的，在平衡状态下人口稀少，并且非常类似于主导自然系综的‘基态’结构。 在我们之前(第一次)NSERC发现基金资助的工作中，我的团队引入了一套质谱学耦合的微流控芯片，可以使用一种名为氢/氢交换的技术来表征“更高能量”的蛋白质构象。在目前的研究计划中，我们将使用这些设备来了解支撑蛋白质功能的动态过程，特别是它们如何实现催化、变构(远距离作用)和致病聚集。例如，在催化的情况下，我们的目标是了解构象动力学如何(或是否)沿着催化反应路径引导酶。为此，我们将表征“正常”酶反应和通过在底物上的关键原子上取代重同位素而被“减缓”的酶反应的动力学(导致“主要动力学同位素效应”)。如果动力学不受反应速率变化的影响，这将表明动力学与催化没有直接联系(或者至少与催化机理中的限速步骤没有联系)。在变构的情况下，我们感兴趣的是了解结构或动力学上的细微变化如何将信息从结合或共价修饰位置‘传递’到蛋白质的远距离部分。了解这一点对于能够预测外围位点的结合、修饰或突变将如何影响蛋白质功能至关重要。在致病聚集的情况下，我们感兴趣的是了解导致弱结构蛋白质(或本质上没有固定结构的无序蛋白质)形成淀粉样变性的构象集合的变化。答案肯定在于“残留结构”的区域，这些区域即使在很大程度上是无序的蛋白质中也存在。我们的快速H/D交换标记技术为我们提供了一种独特的能力来表征内在无序蛋白质(和蛋白质无序区域)中的残基结构，这将使我们能够研究结合或共价修饰驱动的‘无序’系综中的变化如何调节结合特异性和生物活性。 这项拟议的研究的目的正是为了提高我们对蛋白质功能的基本知识。然而，像自然科学中大多数与生物学相关的研究一样，我们希望这些知识最终将为疾病的治疗提供新的途径。我们的见解与构象发病机制以及癌症和神经退行性疾病中蛋白质相互作用网络的崩溃特别相关。

The tools of structural biology (i.e., X-ray crystallography and structural NMR) provide exquisitely detailed 'snapshots' of the 'native structure' of proteins. However, if all proteins were 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. The objective of my group's research is to understand specifically how conformational dynamics drive protein function and, in some cases, the adoption of pathogenic structures that 'clump together' to form dangerous aggregates called amyloids. To do this, we need to know what these higher energy structures look like, but identifying them is no easy task because they tend to be short-lived, weakly populated at equilibrium and very similar to the 'ground-state' structure that dominates the native ensemble. In work funded by our previous (first) NSERC Discovery grant, my group introduced a set of mass spectrometry-coupled microfluidic chips that enable characterization of 'higher energy' protein conformations using a technique called Hydrogen/Deuterium Exchange. In the present research program, we will use these devices to learn about the dynamic processes that underlie protein function, specifically how they enable catalysis, allostery (action at a distance) and pathogenic aggregation. In the case of catalysis, for instance, our aim is to understand how (or if) conformational dynamics guide the enzyme along it's catalytic reaction pathway. To do this, we will characterize dynamics in a 'normal' enzymatic reaction and one that has been 'slowed' by substituting a heavy isotope at a critical atom on the substrate (resulting in a 'primary kinetic isotope effect'). If the dynamics are unaffected by the change in reaction rate, this would indicate that dynamics are not directly linked to catalysis (or at least not to the rate-limiting step in the catalytic mechanism). In the case of allostery, we are interested in learning how subtle changes in structure or dynamics can 'transmit' information from a binding or covalent modification site to distant parts of the protein. Understanding this is crucial for being able to predict how binding, modification or mutation at peripheral sites will influence protein function. In the case of pathogenic aggregation, we are interested in understanding changes in the conformational ensemble that cause weakly structured proteins (or intrinsically disordered proteins, which have essentially no set structure) to be come amyloidogenic. The answer must lie in the regions of 'residual structure' that persist even in proteins that are largely disordered. Our rapid H/D exchange labeling techniques give us a unique capability to characterize residual structure in intrinsically disordered proteins (and disordered regions of proteins), which will allow us to investigate how binding or covalent modification-driven shifts in the 'disordered' ensemble modulate binding specificity and biological activity. The proposed research is aimed squarely at advancing our basic knowledge of protein function. However, like most biologically-linked research in the natural sciences, our hope is that this knowledge will ultimately provide new avenues for the treatment of disease. Our insights are of particular relevance to conformational pathogenesis, and to the breakdown of protein interaction networks in cancer and neurodegenerative disease.