Study of phenotypic and fitness effects of non-functional protein interactions in

描述（由申请人提供）：本研究的总体目标是更好地理解蛋白质的遗传编码物理化学性质的变化如何影响简单细菌有机体的表型变化。具体来说，我们将重点研究拥挤细胞环境中突变引起的非功能性虚假蛋白-蛋白相互作用（NF-PPI）对大肠杆菌细胞内和体外模式生物的表型变异和适应性的影响。这是一项多工具、多尺度的研究，它综合了理论和实验方法来实现其特定目标。理论方法是基于越来越复杂的多尺度模型，其中模型细胞的细胞质以生物物理现实的方式呈现，模型生物的适应度由序列依赖的折叠和模型蛋白质的功能相互作用决定。后者在模型细胞的细胞质中以一定浓度表达，并受到蛋白质质量控制（PQC）的控制。相关实验旨在检验和改进理论模型的主要假设。实验方法是自下而上的，基于合理的基因组编辑，即在基本蛋白质的ORF中感兴趣的突变直接引入大肠杆菌染色体。在体外同时评估突变对受影响蛋白的生物物理和生化特性的影响，在体内对染色体合并相同突变的菌株确定表型和适合度效应。本研究正在解决的问题是：1)代谢酶的不稳定突变在多大程度上引起NF-PPI。2) NF- PPI对突变的适应度和表型效应的贡献有多大，与自关联（聚集）的区别有多大。3) PQC的活性成分-伴侣蛋白和蛋白酶-如何减轻NF-PPI对细菌适应性的潜在有害影响？总的来说，这项研究将极大地促进我们对决定细菌有机体适应性景观的物理化学因素的理解，更好地描述它们的蛋白质组的分子进化和它们在临床环境中对治疗的反应动力学。

DESCRIPTION (provided by applicant): The overarching goal of this research is to achieve a better understanding of how variation of genetically encoded physical chemical properties of proteins affects phenotypic changes of simple bacterial organisms. Specifically, here we focus on the investigation of the impact of non-functional, spurious protein-protein interactions (NF-PPI) caused by mutations in crowded cellular environment on phenotypic variation and fitness of model organisms in silico and in vivo in E. coli cells. This is a multi-tool multiscale research, which synthesizes theoretical and experimental approaches to achieve its Specific Aims. The theoretical approaches are based on multiscale models of increasing complexity where cytoplasm of model cells is presented in a Biophysically realistic manner, and fitness of model organisms is determined by sequence-dependent folding and functional interactions of model proteins. The latter are expressed at certain concentrations in cytoplasm of model cells and are subject to Protein Quality Control (PQC). A related experimentation aims at testing and improving the main assumptions of theoretical models. The experimental approach is bottom up and is based on rational genome editing whereby mutations of interest in ORF of essential proteins are introduced directly on E. coli chromosome. The effect of mutations on Biophysical and Biochemical properties of affected proteins is simultaneously evaluated in vitro and phenotypic and fitness effect is determined in vivo for strains in which same mutations are chromosomally incorporated. The problems that are being addressed in this research are: 1) To what extent do destabilizing mutations in metabolic enzymes give rise to NF-PPI. 2) How much do NF- PPI contribute to fitness and phenotypic effects of mutations and how distinguishable is that from self- association (aggregation). 3) How do active components of PQC - chaperonins and proteases - mitigate potentially detrimental effect of NF-PPI on bacterial fitness? Overall this research will significantly advance our understanding of physical chemical factors that determine fitness landscape of bacterial organisms providing a better description of molecular evolution of their proteomes and the dynamics of their response to treatment in clinical environment.