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整个集合。 因此，蛋白质组可以被视为维持生物体所必需的完整分子机器。 由于任何特定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，地球上曾经存在的所有蛋白质组中所有蛋白质的完整集合构成了理论上可能的序列的极小一部分。因此，尽管经过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得开始探索从未经历过进化的序列成为可能。 拟议的研究旨在设计和生产大量新型蛋白质，这些蛋白质可折叠成明确的 3D 结构，并在生物学相关反应中发挥作用。 这项工作的完成将代表着在开发人工蛋白质组方面取得了重大进展，这种蛋白质组不是由自然进化而来，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·费曼说，“我无法创造的东西，我就不理解。” 因此，新型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：当前的生物技术依赖于从自然界借用的蛋白质序列，而未来的应用将受益于并非自然选择但非常适合工业应用的从头序列。该项目提供了极好的跨学科研究培训机会。 研究人员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（或修改）的序列。 拟议的研究将通过提供数百万种新型蛋白质的库来克服这一限制。这样的集合将比典型的细菌蛋白质组大 1000 倍。 与揭示自然选择的天然蛋白质组研究相比，新近开展的人工蛋白质组研究将拓宽我们对自然进​​化之外的理解，并使实验能够探测在自然界中未观察到的序列、结构和功能。天然生物系统，但仍然可以出现在新型或合成生物学领域。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-折叠蛋白。蛋白质组，无论是天然的还是人造的，都必须包含α和β结构。 -开发和实施用于折叠结构的高通量屏幕。通过对计算设计的蛋白质集合进行可溶且稳定折叠的结构的后续筛选，可以提高蛋白质文库的质量。-确定来自新型蛋白质组的蛋白质的 3 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。-体外有活性、体内有功能的新型蛋白质的分离和进化。 最重要的是，当且仅当新序列的集合包含具有生化活性并提供必要的细胞功能的蛋白质时，它们才会类似于蛋白质组。