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）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”中出现的那样是静态的，那么大多数蛋白质将完全没有功能。为了获得生物活性，蛋白质必须在“天然”结构的集合中获得特定的、更高能量的构象，这些结构是通过被称为构象动力学的热驱动波动填充的。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，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，我们需要知道这些高能量结构是什么样子的，但是识别它们并不是一件容易的事，因为它们往往是短暂的，在平衡状态下人口较少，并且非常类似于主导原生集合的“基态”结构。**在我们之前（第一次）NSERC发现资助的工作中，我的团队引入了一套质谱耦合微流控芯片，可以使用一种称为氢/氘交换的技术来表征“高能量”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，特别是它们如何使催化、变变（远距离作用）和致病性聚集成为可能。以催化为例，我们的目标是了解构象动力学如何（或是否）引导酶沿着催化反应途径进行。为了做到这一点，我们将描述“正常”酶促反应和通过在底物上的关键原子上替换重同位素而“减慢”的酶促反应的动力学特征（导致“初级动力学同位素效应”）。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细微变化如何将信息从结合或共价修饰位点“传递”到蛋白质的遥远部分。了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导致结构弱的蛋白质（或本质上没有固定结构的内在无序蛋白质）成为淀粉样蛋白的构象集合的变化。答案一定在于“残余结构”区域，即使在大部分无序的蛋白质中也存在这些区域。我们的快速H/D交换标记技术使我们具有独特的能力来表征内在无序蛋白质（和蛋白质的无序区域）的残余结构，这将使我们能够研究“无序”集合中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。**拟议的研究完全是为了提高我们对蛋白质功能的基础知识。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将为治疗疾病提供新的途径。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破坏特别相关。

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.