Single Molecule Studies of Protein Folding Mechanisms

描述(申请人提供)：蛋白质折叠成复杂的三维结构的迷人过程在其在细胞和生物体中的各种功能中发挥着关键作用，并在可能导致疾病的错误折叠中发挥关键作用。蛋白质错误折叠和聚集(淀粉样蛋白形成)与几种疾病有关，如帕金森氏症和阿尔茨海默氏症、普里恩病、糖尿病和心脏病。此外，最近的工作支持了一个有趣的想法，即蛋白质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于加深我们对基础生物学和疾病的理解至关重要，了解这一点可能有助于设计这方面的治疗策略。虽然关于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，留下了许多问题有待解决。最近沿着这些思路注意到的一个方面是越来越多的人意识到，一系列蛋白质紊乱(灵活性)在基因组中普遍存在，而且这种紊乱往往与功能和功能障碍密切相关。例如，几种淀粉样蛋白的单体形式具有广泛的无序序列。在这里，我们建议开发和应用新的单分子荧光方法，以进一步加深我们对上述几个方面的理解。我们的工作将集中在与帕金森氏病相关的蛋白α-突触核蛋白上，该蛋白具有多种假定的生物学功能。这种蛋白质作为单体具有很大的无序性，可以在与伴侣结合时折叠，这是它与许多无序蛋白质共享的一个有趣的特征。虽然α-突触核蛋白已经被研究了很多年，但对其复杂而动态的生物物理机制的了解还很有限。通过避免大多数系综实验中固有的平均，我们将探索这个系统及其自然变体在相互作用和聚集过程中的复杂折叠景观，其分辨率通过标准方法是不可行的。我们的创新工作将独特地结合尖端生物物理、光学、生物化学和化学生物学的各个方面。总体而言，这项工作将导致对α-突触核蛋白系统的新水平的理解，并对更好地理解神经退行性疾病的生物学产生影响。此外，我们将开发和实施组合的、最先进的单分子工具，这些工具将广泛应用于许多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.