Toward Artificial Proteomes

走向人工蛋白质组

• 批准号: 1409402
• 负责人: Michael Hecht
• 金额: $ 98.57万
• 依托单位: Princeton University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1409402&HistoricalAwards=false
• 关键词: Toward; Artificial; Proteomes

A proteome can be defined as the entire collection of proteins in an organism. Thus, a proteome can be viewed as the complete set of molecular machines necessary to sustain a living organism. Because many different biochemical functions are required in any particular cell, the proteome of any single organism must include a wide range of proteins with diverse amino acid sequences, three-dimensional structures, and biochemical activities. Nonetheless, the full collection of all proteins in all proteomes that ever existed on earth constitutes a minuscule fraction of the sequences that are theoretically possible. Thus, despite billions of years of evolutionary sampling, the vast majority of sequence space remains unexplored. However, recent advances in synthetic biology, combinatorial methods, and protein design have made it possible to begin exploring sequences that have never been exposed to evolution. The proposed research aims to design and produce large collections of novel proteins that fold into well-defined 3D structures, and function in biologically relevant reactions. Completion of this work will represent a significant advance toward developing artificial proteomes that were not evolved by nature, but nonetheless support the growth of living organisms. The project will have broader significance in basic science and for applied technologies: Richard Feynman said, "What I cannot create, I do not understand." Thus, the creation of novel proteins will both test our knowledge, and enhance our understanding of protein biochemistry, biophysics, and molecular evolution. Design and construction of artificial proteomes will also impact applied science and biotechnology: Current biotechnology relies on protein sequences borrowed from nature, while future applications will benefit from de novo sequences that were not selected by nature, but are well-suited for industrial applications. This project provides excellent interdisciplinary research training opportunities. The investigator will present this work also to the public and discuss it in the classroom.Previous studies of proteins and proteomes were limited to sequences isolated (or modified) from natural systems. The proposed research will overcome this limitation by making available libraries of millions of novel proteins. Such collections will be 1000-fold larger than typical bacterial proteomes. In contrast to studies of natural proteomes, which reveal what was selected by nature, newly enabled studies of artificial proteomes will broaden our understanding beyond what evolved in nature, and will enable experiments that probe sequences, structures, and functions, which are not observed in natural biological systems, but nonetheless can occur in the realm of novel or synthetic biologies. The research will harness both combinatorial/ experimental and computational/theoretical approaches to pursue the following aims:-Design and construction of large collections of novel alpha-helical proteins.-Design and construction of large collections of novel beta-sheet proteins. Proteomes, whether natural or artificial, must contain both alpha and beta structures. -Development and implementation of a high throughput screen for folded structures. The quality of protein libraries will be enhanced by subjecting collections of computationally designed proteins to follow-up screens for structures that are soluble and stably folded.-Determination of 3-dimensional structures and stabilities of proteins from the novel proteome. Successful designs will produce sequences that fold into expected structures.-Isolation and evolution of novel proteins that are active in vitro and functional in vivo. Most importantly, collections of novel sequences will resemble proteomes if and only if they include proteins that are biochemically active and provide essential cellular functions.

蛋白质组可以定义为生物体中蛋白质的全部集合。因此，蛋白质组可以被看作是维持一个生物体所必需的一整套分子机器。由于任何特定的细胞都需要许多不同的生化功能，因此任何单一生物体的蛋白质组必须包括具有不同氨基酸序列、三维结构和生化活性的广泛蛋白质。尽管如此，地球上曾经存在过的所有蛋白质组中所有蛋白质的完整集合只占理论上可能的序列的极小部分。因此，尽管有数十亿年的进化采样，绝大多数序列空间仍未被探索。然而，合成生物学、组合方法和蛋白质设计的最新进展使探索从未暴露于进化的序列成为可能。拟议的研究旨在设计和生产大量的新型蛋白质，这些蛋白质折叠成明确的3D结构，并在生物相关反应中发挥作用。这项工作的完成将代表着开发人工蛋白质组的重大进步，这些蛋白质组不是自然进化的，但仍然支持生物体的生长。该项目将在基础科学和应用技术方面具有更广泛的意义：理查德·费曼（Richard Feynman）曾说过：“我不能创造的东西，我就不理解。”因此，新蛋白质的创造将考验我们的知识，并增强我们对蛋白质生物化学、生物物理学和分子进化的理解。人工蛋白质组的设计和构建也将影响应用科学和生物技术：目前的生物技术依赖于从自然界借来的蛋白质序列，而未来的应用将受益于那些不是自然选择的、但非常适合工业应用的新序列。本项目提供优秀的跨学科研究培训机会。研究者还将向公众展示这项工作，并在课堂上进行讨论。以前对蛋白质和蛋白质组的研究仅限于从自然系统中分离（或修饰）的序列。拟议的研究将通过提供数百万种新蛋白质的文库来克服这一限制。这样的集合将比典型细菌蛋白质组大1000倍。与揭示自然选择的天然蛋白质组学研究相反，人工蛋白质组学的新研究将拓宽我们对自然界进化之外的理解，并将使实验能够探测在自然生物系统中未观察到的序列、结构和功能，但仍然可以发生在新生物或合成生物学领域。该研究将利用组合/实验和计算/理论方法来实现以下目标：-设计和构建大量新型α -螺旋蛋白。-设计和构建大量新型β -片蛋白。蛋白质组，无论是天然的还是人工的，都必须同时包含α和β结构。-开发和实现折叠结构的高通量筛。通过将计算设计的蛋白质集合进行后续筛选，以寻找可溶和稳定折叠的结构，将提高蛋白质文库的质量。-测定新蛋白质组蛋白的三维结构和稳定性。成功的设计将产生折叠成预期结构的序列。-体外活性和体内功能的新蛋白的分离和进化。最重要的是，新序列的集合将类似于蛋白质组，当且仅当它们包含具有生物化学活性并提供基本细胞功能的蛋白质时。