The role of the free energy landscape in Parkin's function and dysfunction in health and disease

环泛素E3连接酶是一个蛋白质超家族，对蛋白质的动态平衡和信号转导起关键作用。 真核生物。E3连接酶功能障碍与无数人类疾病有关。这项提案将重点放在 泛素E3连接酶Parkin的调节。帕金是有控制地摧毁 线粒体因自噬(有丝分裂)而受损。受控的有丝分裂吞噬对于心脏来说尤其重要 和神经元健康。Parkin基因突变引起的不受控制的有丝分裂吞噬现象显然是早发的原因 帕金森氏病(EoPD)。帕金现在与其他一些神经疾病有牵连， 心肌病和各种癌症。这里的中心目标是创建一种对物理的理解 调节帕金森氏症的基础以及临床观察到的突变如何促进未受调节的活动导致 未充分控制有丝分裂和其他生物缺陷。 虽然人们对帕金功能的生物学和结构基础了解很多，但对它的确定却很少 其监管的物质基础。Parkin活性被其分子内与一个 泛素样结构域，由磷酸化泛素(PUB)结合变构激活。 Ubl结构域的磷酸化也促进了激活。这一复杂的监管交集 机理只能通过对基本热力学的严格剖析才能理解。如果没有 这种知识不能完全解释导致疾病的突变的影响。 我们将利用帕金生物学和结构基础的广泛知识基础 它的功能，以解决鲜为人知的热力学的变构调节帕金。其基础是 对于调节控制的帕金将在现代统计热力学中进行描述 蛋白质组。变构调节剂和翻译后修饰的影响将是 通过质谱学和核磁共振谱监测的综合氢交换进行检测； 先进的核磁共振弛豫技术；单分子荧光；量热法；酶学；以及 诱变。 对帕金规则的更严格和更完整的理解将使强有力的解释成为可能 病理性突变。并不是所有的病理性突变都可以简单地解释为 直接影响催化部位的蛋白质水平或突变。常见病理例证 将对突变进行研究，以揭示它们对帕金监管忠诚度的影响的基础，具有更长的- 确定如何通过小分子干预减轻这种影响的范围目标。

The RING ubiquitin E3 ligases are a superfamily of proteins critical to protein homeostasis and signaling in eukaryotes. Dysfunctions in E3 ligases are implicated in innumerable human diseases. This proposal focuses on the regulation of the ubiquitin E3 ligase Parkin. Parkin is central to the controlled destruction of damaged mitochondria by autophagy (mitophagy). Controlled mitophagy is particularly essential to cardiac and neuronal health. Uncontrolled mitophagy due to mutations in Parkin is clearly a driver of early onset Parkinson's disease (eoPD). Parkin is now implicated in a number of other neurological diseases, cardiomyopathy and in various cancers. The central goal here is to create an understanding the physical basis for regulation of Parkin and how clinically observed mutations promote unregulated activity leading to inadequately controlled mitophagy and other biological defects. Though much is known about the biology and structural basis of Parkin function, very little is certain about the physical basis for its regulation. Parkin activity is suppressed by its intra-molecular association with a ubiquitin-like domain and is allosterically activated by the binding of phosphorylated ubiquitin (pUb). Phosphorlyation of the Ubl domain also promotes activation. This complicated intersection of regulatory mechanisms can only be understood by the rigorous dissection of the underlying thermodynamics. Without this knowledge one cannot fully interpret the effects of mutations that lead to disease. We shall take advantage of the broad foundation of knowledge of the biology of Parkin and structural basis of its function to address the poorly understood thermodynamics of allosteric regulation of Parkin. The basis for regulatory control of Parkin will be cast in a modern statistical thermodynamics description of the protein ensemble. The influence of allosteric regulators and post-translational modifications will be examined by comprehensive hydrogen exchange monitored by mass spectrometry and NMR spectroscopy; advanced NMR relaxation techniques; single molecule fluorescence; calorimetry; enzymology; and mutagenesis. A more rigorous and complete understanding of the regulation of Parkin will enable a robust interpretation of pathological mutations. Not all pathological mutations can be simply explained as mutations that disrupt the levels of protein or mutations that directly impact the catalytic site. Examples of common pathological mutations will be examined to reveal the basis for their effects on Parkin's regulatory fidelity, with a longer- range goal of determining how this impact might be mitigated by small molecule intervention.