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.
. PI: Lakshmi Pulakat Biological nitrogen fixation by nitrogenase is an important process that helps to convert atmospheric nitrogen into a usable form for plants. NifH is a component of Nitrogenase that performs biological nitrogen fixation. Previous studies have shown that NifH cannot function in the absence of an essential accessory protein, NifM. Therefore, expression of functional NifH in other systems requires co-expression of NifM. We identified that NifM is a Peptidyl-Prolyl cis/trans Isomerase (PPIase) and its PPIase activity is needed for generating functional NifM. Peptidyl-prolyl cis/trans isomerases (PPIases) are chaperone proteins that play a pivotal role in the folding of many prokaryotic and eukaryotic proteins that play critical roles in a variety of biological functions. PPIases catalyze the cis-trans isomerization of proline peptide bonds in their target, or substrate proteins. Inappropriate PPIase-substrate interactions can lead to misfolding of substrate proteins and inhibition of biological functions that can lead to cell death and pathogenesis. Therefore, understanding the specifics of PPIase-substrate interactions that lead to proper folding versus misfolding will provide us new tools to regulate protein functions. We developed a prokaryotic genetic system in which we could systematically analyze the effect of a PPIase (NifM), on two structurally-similar substrate proteins (NifH and ChlL, a protein required for light-independent chlorophyll biosynthesis). Making this system even more unique is our observation that the PPIase (NifM) exerts opposing effects on the two substrates – it makes NifH functional, but inhibits the function of ChlL. Therefore, this system probes 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. An important outcome of our studies is that we have generated a library of mutant NifH proteins by using a DNA shuffling method or mutagenesis. Then we used our system to probe the ability of these mutants to function independent of NifM. Through this analysis we identified some of the proline residues that serve as substrates for NifM and found that replacement of these proline residues could render functional NifH that does not require NifM. We have also identified different structural changes that can lead to the formation of NifM-independent NifH. Thus a significant outcome of these studies is that we have identified ways to reduce the need for an additional accessory protein, NifM, to generate a functional nitrogenase in different systems. ChlL is not the natural substrate for NifM. We uncovered that the region of ChlL that interacts with NifM is structurally similar to the NifM-interacting region of the NifH. Moreover, we generated a NifH-ChlL chimeric protein that could support biological nitrogen fixation in the absence of NifM. Our observations suggest that NifM could have contributed to the functional divergence of biological nitrogen fixation and photosynthesis during evolution by virtue of its ability to exert opposing effects on structurally similar substrates, ChlL and NifH. The scientific broader impact of this project is the elucidation of the mode of action of a large variety of PPIases across the species, including human PIN1, and P. aeruginosa protein NarM. Furthermore, the infrastructure developed with the help of NSF funding was instrumental in our studies to elucidate the roles of two other proteins, (ORF9 and ORF10) in the functions of Nitrogenase complex. Finally, another important broader impact of this project is that it helped to sustain ongoing graduate and undergraduate training in modern molecular and cellular biology that includes, but not limited to areas such as bioinformatics, molecular modeling, genetics and microbiology at University of Missouri-Columbia and Mississippi State University. This NSF funding provided research experience and training to 4 graduate students, 15 undergraduate students, 3 post-doctoral fellows and 3 high school students. The poster entitled "Role of NarM, a novel PPIase in the anaerobic growth of biofilm forming pathogen Pseudomonas aeruginosa" received the first place in the 2013 poster competition held by Missouri Foundation for Medical Research on the Harry S. Truman Memorial Veterans Affairs Hospital Research Day. Two post-doctoral fellows (Drs. Koppula and Somashekara), one former graduate student (Dr. Raja) and one undergraduate student (Mr. Dudeja) who received training from this NSF-supported research are co-authors on this poster. NSF supported training helped two of our post-doctoral fellows to obtain faculty positions and graduate students to take up post-doctoral positions or continue their career in other science education related jobs (Research Associate in Media & Communication at UNC Greensboro). Undergraduate students who have completed their degrees during the NSF funding period have joined either MD (UM-Columbia School of Medicine) or DPM (Scholl College of Podiatric Medicine-Chicago) programs.
