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.
