Toward Artificial Proteomes

蛋白质组可以被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一个活的有机体所必需的一整套分子机器。因为任何特定的细胞都需要许多不同的生化功能，所以任何单个生物体的蛋白质组都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的蛋白质。尽管如此，地球上曾经存在的所有蛋白质组中的所有蛋白质的全部收集只构成了理论上可能的序列的一小部分。因此，尽管进行了数十亿年的进化采样，但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人们有可能开始探索从未经历过进化的序列。这项拟议的研究旨在设计和生产大量新的蛋白质，这些蛋白质折叠成定义明确的3D结构，并在生物相关的反应中发挥作用。这项工作的完成将代表着朝着开发人造蛋白质组的方向迈进了一大步，这些蛋白质组不是自然进化的，但仍然支持活的有机体的生长。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：“我不能创造的东西，我不理解。”因此，新蛋白质的产生既考验了我们的知识，又增强了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借用自然的蛋白质序列，而未来的应用将受益于非自然选择的从头序列，但非常适合工业应用。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介绍这项工作，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。这项拟议的研究将通过建立数百万种新蛋白质的可用文库来克服这一限制。这样的收集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反，新启用的人工蛋白质组研究将拓宽我们对自然进化的理解，并将使探索序列、结构和功能的实验成为可能，这些序列、结构和功能在自然生物系统中没有观察到，但仍然可以在新的或合成生物学领域出现。这项研究将利用组合/实验和计算/理论方法来追求以下目标：-设计和构建大量新型α-螺旋蛋白质。-设计和构建大量新型β-折叠蛋白质。蛋白质组，无论是天然的还是人造的，都必须同时包含α和β结构。-开发和实施用于折叠结构的高通量屏幕。蛋白质文库的质量将通过对计算机设计的蛋白质集合进行后续筛选来提高，以寻找可溶和稳定折叠的结构。-从新的蛋白质组确定蛋白质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。-分离和进化在体外具有活性和在体内具有功能的新蛋白质。最重要的是，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，新序列的集合才与蛋白质组相似。

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.