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

肽基脯氨酰顺反异构酶（PPIases）是催化底物蛋白中脯氨酸肽键顺反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PPIases的爆炸式增长。许多原核和真核蛋白质依赖于其同源PPIase来实现其功能形式。然而，它们与结构上类似于其天然底物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解开异常的PPIase底物相互作用，导致蛋白质的错误折叠。 该研究方法采用了一个模型系统，该系统由原核PPIase酶NifM和两种结构相似的底物蛋白NifH（细菌蛋白）和ChlL（叶绿体蛋白）组成。 该项目中采用的实验系统将探讨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。工作的合理性在于，由于NifM通过其PPIase活性在其底物的某些区域上发挥其作用，因此必须有可能通过相应底物的特定残基/区域的突变来使NifM对NifH不依赖，并且使NifM对ChlL耐受。 虽然许多PPIase已经在物种间进行了表征，但迄今为止，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（NifM独立的nifH和NifM耐受chIL）和嵌合蛋白。 对这些突变体的分子分析将解释是什么样的变化使这些蛋白质免受PPI酶的影响，这些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ChlL在叶绿素生物合成中的功能。更广泛的影响：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，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，了解参与NifH辅助蛋白介导的活化的分子机制将推进一般的金属酶的功能组装领域。 该项目的教育影响是，它将维持在MSU现代分子和细胞生物学正在进行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。学生将在两种环境中获得学术学分的研究经验，一种是课堂环境，另一种是作为独立研究人员。

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.