Genetic Analysis of the Differential Effects of NifM, a PPIase, on its Substrates

肽基-脯氨酸顺式/反式异构酶（PPIases）是催化脯氨酸肽键在其底物蛋白中的顺式-反式异构化并调节其折叠的酶。使用生物信息学工具的基因组测序和分析已经导致跨物种发现新的PPIases的爆炸式增长。许多原核和真核蛋白质依赖于它们的同源PPIases来实现它们的功能形式。然而，它们与结构上与其天然底物相似的蛋白质相互作用的能力可能导致这些蛋白质的错误折叠并阻碍其功能特性。这些酶如何改变其底物蛋白的功能仍然是一个谜。该项目的重点是开发创新的方法来揭示导致蛋白质错误折叠的异常ppase -底物相互作用。该研究方法采用了一个由原核PPIase酶NifM和两种结构相似的底物蛋白（细菌蛋白NifH和叶绿体蛋白ChlL）组成的模型系统。该项目中采用的实验系统将探索PPIases作为分子开关的潜在有益或有害能力，这些分子开关可以激活一种蛋白质，但抑制其他结构相似的蛋白质。这项工作的理由是，由于NifM通过其PPIase活性在其底物的某些区域发挥作用，因此必须有可能通过各自底物的特定残基/区域的突变，使NifM对NifH不依赖，并使NifM对ChlL耐受。尽管许多ppiase已经在物种间被表征，但迄今为止，还没有关于ppiase不依赖或ppiase耐受的底物突变体的报道。该项目涉及这些突变体（不依赖nifm的nifH和耐nifm的chlL）和嵌合蛋白。对这些突变体的分子分析将解释是什么变化使这些蛋白质免受ppase的影响，这一信息将对目前对蛋白质折叠途径机制的理解产生重大影响。这项工作将确定nifm独立的NifH是否能够补充chl在叶绿素生物合成中的功能。广泛影响：NifH是一种复杂的金属酶，与atp酶和其他几种金属蛋白（如CompA和MinD）具有很强的结构同源性，它们分别在谷氨酸降解和细胞分裂的空间调节中起作用。因此，了解NifH辅助蛋白介导的激活的分子机制将促进金属酶功能组装领域的发展。这个项目对教育的影响是，它将支持密歇根州立大学正在进行的现代分子和细胞生物学的研究生和本科生培训。本项目所采用的实验系统将成为生物学领域的优秀教学工具，包括但不限于遗传学、细胞与分子生物学、分子建模和生物信息学。学生将在两种情况下获得学术学分的研究经验，在课堂环境和作为独立研究者。

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that catalyze the cis-trans isomerization of proline peptide bonds in their substrate proteins and regulate their folding. Genomic sequencing and analyses using bioinformatic tools have led to an explosion of discovery of new PPIases across species. Many prokaryotic and eukaryotic proteins are dependent on their cognate PPIases for achieving their functional form. However, their ability to interact with proteins that are structurally similar to their natural substrates can contribute to misfolding of these proteins and hinder their functional properties. How these enzymes alter the functionality of their substrate proteins is still an enigmatic topic. The project focus is to develop innovative approaches to unravel abnormal PPIase-substrate interactions that lead to the misfolding of proteins. The research approach employs a model system that consists of a prokaryotic PPIase enzyme, the NifM, and two structurally similar substrate proteins, the NifH, a bacterial protein, and the ChlL, a chloroplast protein. The experimental system employed in the project will probe into the potentially beneficial or detrimental ability of PPIases to act as molecular switches that can activate one protein but inhibit other structurally-similar proteins. The rational of the work is that since NifM exerts its effects through its PPIase activity on certain regions of its substrates, it must be possible to render NifM-independence to NifH and NifM-tolerance to ChlL through the mutation of specific residues/regions of the respective substrates. Although many PPIases have been characterized across species, to date, there are no reports on PPIase-independent, or PPIase-tolerant mutants of their substrates. The project involves such mutants (NifM-independent nifH and NifM-tolerant chlL) and chimeric proteins. Molecular analyses of these mutants will explain what changes free these proteins from PPIase-influence, and this information will have a major impact on the current understanding of the mechanisms of protein folding pathways. The work will determine whether the NifM-independent NifH is capable of complementing the functions of ChlL in chlorophyll biosynthesis.Broader impacts: NifH is a complex metalloenzyme that shares strong structural homology with ATPases and several other metalloproteins such as CompA and MinD, which function in glutamate degradation and the spatial regulation of cell division, respectively. Thus, understanding the molecular mechanisms involved in NifH accessory protein-mediated activation will advance the fields of the functional assembly of metalloenzymes in general. The educational impact of this project is that it will sustain the ongoing graduate and undergraduate training in modern molecular and cellular biology at MSU. The experiment system that will be employed in the project will serve as an excellent teaching tool in the fields of biology including but not limited to genetics, cellular and molecular biology, molecular modeling, and bioinformatics. Students will be provided research experience for academic credit in two settings, in a classroom setting and as an independent researcher.