PROTstretch - Dynamic structure of a nanomachine involved in proteome quality control: a combined NMR/SAXS/SANS study of the PAN unfoldase

该项目的目的是深入了解一类重要的生物大分子机器的分子作用模式，即所谓的AAA atp酶展开机器。一个健康的蛋白质组，即在细胞中给定时间点存在的蛋白质集合，对于任何生物体的正常功能都是必不可少的。在基因表达和翻译后水平上存在许多调节机制，这些机制控制着蛋白质的数量、特异性和活性，以响应可变的内外环境。在活细胞中，这些机制之一包括捕获和降解失能蛋白。这种功能是至关重要的，因为异常折叠的蛋白质有聚集的倾向，并可能对细胞造成不可逆转的损害。特定蛋白质降解的核心挑战是完成显示异常构象状态或不再需要的蛋白质的展开。这项任务是由不同类型的AAA atp酶展开机器（展开酶）完成的，这些机器准备通过蛋白酶体降解蛋白质。由于其在蛋白质降解途径中的关键地位，unfoldases目前被讨论为治疗干预的潜在先导化合物。在人类中，有缺陷的蛋白质消除功能的改变会导致许多破坏性疾病，如阿尔茨海默病或亨廷顿病，也是感染性疾病的基础，如朊病毒。特别是神经退行性疾病，更有可能随着年龄的增长而发生，这对世界老龄化人口构成了一个相当大的健康问题。在这种情况下，深入了解展开酶机制的作用模式是生物医学的一个主要目标。经典的结构生物学技术，如晶体学，在应用于如此复杂的动态和寡聚系统时是有限的，并且不能提供对其作用模式的充分理解。在这里，我们使用结构生物学技术的强大组合来解决这些问题：小角度x射线（SAXS）和中子（SANS）散射以及核磁共振波谱（NMR）。我们建议将这种技术组合应用于使用GFP作为衬底的PAN系统。

The aim of the p project is to gain insight into the molecular mode of action of an important class of biomacromolecular machines, the so-called AAA ATPase unfolding machines. A healthy proteome, i.e. the ensemble of proteins present at a given point in time in a cell, is essential for the correct functioning of any organism. Numerous regulatory mechanisms exist at the gene expression and post-translational levels that control the amount, specificity and activity of proteins, in response to variable internal and external environments. In living cells one of these mechanisms consists in trapping and degrading disabled proteins. This function is critical, since abnormally folded proteins have a tendency to aggregate and can provoke irreversible damages to the cell. The central challenge for specific protein degradation is to complete the unfolding of proteins that display an abnormal conformational state or that are no longer needed. This task is accomplished by different classes of AAA ATPases unfolding machines (unfoldases) that prepare the proteins for degradation via the proteasomes. Because of their pivotal position in the protein degradation pathway, unfoldases are currently discussed as potential lead compounds for therapeutic intervention. In humans, an altered function in defective protein elimination can cause a number of destructive diseases, such as Alzheimer or Huntington, and is also at the basis of infective diseases, such as prions. Neurodegenerative diseases, in particular, are more likely to occur with age, which constitutes a considerable health issue for the aging world population. In this context, understanding in depth the mode of action of unfoldase machineries represents a major goal in biomedicine. Classical structural biology techniques, such as crystallography, are limiting when applied to such complex dynamic and oligomeric systems and cannot provide a full understanding of their mode of action. Here we address these questions using a powerful combination of structural biology techniques in solution: small-angle X-ray (SAXS) and neutron (SANS) scattering as well as nuclear magnetic resonance spectroscopy (NMR). We propose to apply this combination of techniques to the PAN system using GFP as a substrate.