Single Molecule Studies of Protein Folding Mechanisms

描述（由申请人提供）：蛋白质折叠成复杂的三维结构的迷人过程在细胞和生物体的无数功能中起着关键作用，在可能导致疾病的错误折叠中起着关键作用。蛋白质错误折叠和聚集（淀粉样蛋白形成）与多种疾病有关，如帕金森病和阿尔茨海默病、朊病毒病、糖尿病和心脏病。此外，最近的研究支持了一个有趣的观点，即淀粉样蛋白也可以在生物学中发挥功能作用。因此，了解蛋白质折叠的机制对我们进一步了解基础生物学和疾病至关重要，了解这些机制可以帮助我们在这方面设计治疗策略。虽然我们对蛋白质折叠和聚集已经了解了很多，但这些都是极其复杂的过程，留下了许多有待解决的问题。沿着这些路线最近注意到的一个方面是越来越多的人认识到蛋白质紊乱（灵活性）的频谱在基因组中普遍存在，并且这种紊乱通常与功能和故障密切相关。例如，几种淀粉样蛋白的单体形式具有广泛的无序序列延伸。在这里，我们建议开发和应用新的单分子荧光方法，以进一步了解上述几个方面的下一步关键步骤。我们的工作将集中在帕金森病相关蛋白α-突触核蛋白上，它具有多种假定的生物学功能。这种蛋白质作为一个单体具有严重的无序性，并且在与伴侣结合时可以折叠，这是它与许多无序蛋白质共有的一个有趣的特征。尽管对α-突触核蛋白的研究已经进行了多年，但对其复杂的动态生物物理特性的了解有限。通过避免大多数集成实验中固有的平均，我们将以标准方法无法实现的分辨率探索该系统在相互作用和聚合过程中的复杂折叠景观及其自然变体。我们的创新工作将独特地结合前沿生物物理学、光学、生物化学和化学生物学的各个方面。总的来说，这项工作将导致对α-突触核蛋白系统的认识达到一个新的水平，对更好地理解神经退行性疾病的生物学具有重要意义。此外，我们将开发和实施组合，最先进的单分子工具，这将广泛适用于其他生物重要分子的研究。

DESCRIPTION (provided by applicant): The fascinating process by which proteins fold to complex 3-dimensional structures plays critical roles in their myriad functions in cells and organisms, and in misfolding that can lead to disease. Protein misfolding and aggregation (amyloid formation) are implicated in several diseases such as Parkinson's and Alzheimer's diseases, prion diseases, diabetes, and heart disease. Furthermore, recent work supports the intriguing idea that protein amyloids can also have functional roles in biology. Therefore, understanding the mechanisms by which proteins fold is critical to furthering our understanding of basic biology and disease, understanding that could later assist with design of therapeutic strategies in this regard. While much has been learned about protein folding and aggregation, these are extremely complex processes, leaving a host of issues yet to be resolved. One aspect of recent note along these lines is the increasing realization that a spectrum of protein disorder (flexibility) is prevalent in genomes, and that this disorder is often intimately linked to functio and malfunction. For example, monomeric forms of several amyloid-forming proteins have extensive stretches of disordered sequence. Here, we propose to develop and apply novel single-molecule fluorescence methods to take next key steps in furthering our understanding of several of the above aspects. Our work will focus on the Parkinson's disease-linked protein α-synuclein, which has multiple putative biological functions. This protein has substantial disorder as a monomer and can fold upon binding to partners, an interesting feature that it shares with many disordered proteins. Although α-synuclein has been investigated for many years, limited insight has been gained about its complex and dynamic biophysics. By avoiding the averaging inherent in most ensemble experiments, we will probe the complex folding landscape of this system and its natural variants during interactions and aggregation at a resolution not feasible by standard methods. Our innovative work will uniquely combine facets of cutting-edge biophysics, optics, biochemistry, and chemical biology. Overall, the work will result in a new level of understanding of the α-synuclein system, with implications for better understanding of the biology of neurodegenerative diseases. Furthermore, we will develop and implement combination, state-of-the-art single-molecule tools which will be broadly applicable in studies of a host of other biologically important molecules.