Computation-Guided Protein Recombination and Evolution

描述（由申请人提供）：基因外显子理论假设古代基因组具有内含子-外显子结构，通过外显子的重组允许蛋白质结构和功能的快速多样化。虽然许多研究已经调查了哪些现代外显子对应于蛋白质的结构单元，并且结构模型已经确定了在非同源蛋白质中普遍存在的假定的蛋白质构建块，但控制多肽是否可以在不同蛋白质之间交换的原理仍然不清楚。我们已经开发了一种算法，叫做SCHEMA，来预测蛋白质的哪些元素，或SCHEMA，可以在不破坏折叠结构的情况下在同源蛋白质之间交换。我们建议将生化和计算研究结合起来，使用SCHEMA和其他新颖的算法，其目标是阐明通过重组非破坏性重组和新功能进化的规则。我们的具体目标是：1)确定内酰胺酶和细胞色素P450单加氧酶同源重组时可耐受结构破坏的模式预测阈值；2)开发预测高效重组适应度搜索的新算法；3)通过内酰胺酶和细胞色素p450的实验室进化来表征预测搜索策略的有效性；4)重组结构破坏预测优化；5)研究非同源蛋白是否可以重组生成折叠蛋白，使用算法来引导交叉位置。这些研究应该允许我们发现同源和非同源重组何时保护蛋白质结构，并扩展我们对进化如何探索序列，结构和功能多样性的理解。此外，这些研究应该通过实验室进化为蛋白质工程提供新的工具，并在生物医学上应用于开发新的生物材料、生物传感器、催化剂和基于蛋白质的疗法。

DESCRIPTION (provided by applicant): The Exon Theory of Genes hypothesizes that ancient genomes had an intron-exon structure that allowed for rapid diversification of protein structure and function through recombination of exons. While much research has investigated which modern exons correspond to structural units in proteins, and structural models have identified putative protein building blocks ubiquitous among non-homologous proteins, the principles that govern whether a polypeptide can be exchanged among different proteins remain unclear. We have developed an algorithm, called SCHEMA, to predict what elements, or schemata, of a protein can be swapped among homologous proteins without disrupting the folded structure. We propose a combination of biochemical and computational studies using SCHEMA and other novel algorithms, whose goals are to elucidate the rules governing non-disruptive recombination and evolution of novel functions by recombination. Our specific aims are to: 1) determine the SCHEMA-predicted threshold(s) of tolerable structural disruption upon homologous recombination for lactamases and cytochrome P450 monooxygenases; 2) develop novel algorithms for predicting efficient recombination fitness searches; 3) characterize the effectiveness of predicted search strategies through laboratory evolution of lactamases and cytochrome P450s; 4) optimize predictions of recombinant structural disruption; and 5) investigate if nonhomologous proteins can be recombined to generate folded proteins, using the algorithms to guide crossover locations. These studies should allow us to discover when homologous and non-homologous recombination conserves protein structure and expand our understanding of how evolution explores sequence, structural, and functional diversity. Furthermore, these studies should generate new tools for protein engineering by laboratory evolution, with biomedical applications in the development of new biomaterials, biosensors, catalysts, and protein-based therapeutics.