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，该结构允许通过外显子重组实现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中的结构单元，并且结构模型已经确定了在非同源蛋白质中普遍存在的推定蛋白质构建块，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称为SCHEMA的算法，用于预测蛋白质的哪些元素或图式可以在同源蛋白质之间交换而不破坏折叠结构。我们提出了一个组合的生物化学和计算研究，使用SCHEMA和其他新的算法，其目标是阐明规则的非破坏性重组和进化的新功能重组。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容忍的结构破坏的SCHEMA预测阈值; 2）开发用于预测有效重组适合度搜索的新算法; 3）通过内酰胺酶和细胞色素P450的实验室进化表征预测搜索策略的有效性; 4）优化重组结构破坏的预测;以及5）研究非同源蛋白质是否可以重组以产生折叠蛋白质，使用算法来指导交叉位置。这些研究将使我们能够发现同源和非同源重组何时保存蛋白质结构，并扩大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产生蛋白质工程的新工具，在开发新的生物材料，生物传感器，催化剂和基于蛋白质的治疗方法中具有生物医学应用。

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.