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

肽基-脯氨酰顺/反式异构酶(PPIase)是催化其底物蛋白质中的脯氨酸肽键的顺反异构化并调节其折叠的酶。基因组测序和使用生物信息学工具的分析导致了跨物种发现新的PPIase的爆炸性增长。许多原核生物和真核生物的蛋白质依赖于它们的同源PPIase来实现其功能形式。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会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质的功能仍然是一个谜。该项目的重点是开发创新的方法来解开导致蛋白质错误折叠的PPIase-底物的异常相互作用。该研究方法采用了一个由原核生物PPIase酶NifM和两个结构相似的底物蛋白NifH和ChlL组成的模型系统。该项目采用的实验系统将探索PPIase作为分子开关的潜在有利或有害能力，这种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。这项工作的原理是，由于NifM是通过其底物某些区域的PPIase活性来发挥作用的，因此必须有可能通过突变相应底物的特定残基/区域来使NifM独立于nifH，并使NifM对ChlL耐受。虽然许多PPIase已经跨物种被鉴定，但到目前为止，还没有关于其底物的PPIase非依赖性或PPIase耐受性突变体的报道。该项目涉及这样的突变体(非NifM依赖的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不受PPIase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。这项工作将确定不依赖于NifM的NifH是否能够补充ChlL在叶绿素生物合成中的功能。间接影响：NifH是一种复杂的金属酶，与ATPase和其他几种金属蛋白如CompA和Mind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发挥作用。因此，了解NifH辅助蛋白介导的激活的分子机制将推动金属酶功能组装领域的发展。这个项目的教育影响是，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。将在该项目中使用的实验系统将作为生物学领域的优秀教学工具，包括但不限于遗传学、细胞和分子生物学、分子建模和生物信息学。学生将在两个环境中为学术学分提供研究经验，在课堂环境中和作为独立研究员。

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.