Conformational Disorder in Protein Function and Pathogenic Aggregation

结构生物学的工具（即，X射线晶体学和结构核磁共振（NMR）提供了蛋白质“天然结构”的精致详细的“快照”。然而，如果所有的蛋白质都像它们在这些“快照”中出现的那样静止，那么大多数蛋白质将完全没有功能。为了实现生物活性，蛋白质必须在“类天然”结构的集合中获得特定的更高能量的构象，这些结构通过称为构象动力学的热驱动波动来填充。我的团队的研究目标是具体了解构象动力学如何驱动蛋白质功能，以及在某些情况下，采用致病结构“聚集在一起”形成称为淀粉样蛋白的危险聚集体。要做到这一点，我们需要知道这些更高能量的结构是什么样子的，但识别它们并不容易，因为它们往往是短暂的，在平衡状态下的弱填充，并且非常类似于主导原生系综的“基态”结构。我的小组介绍了一套质谱联用微流控芯片，可以使用一种称为氢/氘交换的技术来表征“高能”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）和致病性聚集。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）引导酶沿着它的催化反应途径。要做到这一点，我们将在一个“正常”的酶促反应和一个已被“放缓”取代的重同位素在基板上的关键原子（导致“主要动力学同位素效应”）的动态特性。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作用没有直接联系（或者至少与催化机制中的限速步骤没有直接联系）。在变构的情况下，我们感兴趣的是了解结构或动力学的微妙变化如何将信息从结合或共价修饰位点“传递”到蛋白质的远处部分。理解这一点对于能够预测外周位点的结合、修饰或突变如何影响蛋白质功能至关重要。在致病性聚集的情况下，我们感兴趣的是了解构象系综的变化，导致弱结构蛋白质（或本质上无序的蛋白质，基本上没有固定的结构）成为淀粉样蛋白。答案一定在于“残余结构”的区域，即使在很大程度上是无序的蛋白质中也会存在。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（和蛋白质的无序区域）中的残留结构，这将使我们能够研究“无序”系综中的结合或共价修饰驱动的转变如何调节结合特异性和生物活性。这项拟议中的研究旨在提高我们对蛋白质功能的基本认识。然而，就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能为疾病的治疗提供新的途径。我们的见解是特别相关的构象发病机制，并在癌症和神经退行性疾病的蛋白质相互作用网络的崩溃。

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.