Progress 06/15/21 to 11/06/23
Outputs
Target Audience:Throughout the duration of this project, I reached the research scientist and undergraduate student subsets of our target audience through sharing the work we've conducted in this project at scientific conferences and the publication of a manuscript in PNAS. I presented a poster detailing our progress on this project at the 2022 and 2023 WSU-NIH Protein Biotechnology Training Program Symposia, which feature industry speakers and are attended primarily by WSU graduate students and professors, and I used the support of this project to attend the 25th North American Symbiotic Nitrogen Fixation Conference held in Madison, WI, where I reached industry and academic research scientists as well as graduate and undergraduate students that are working on various aspects of biological nitrogen fixation. Additionally, the work in this project was featured in a Futurum Careers article targeted at high-school-age students. Changes/Problems:Following acceptance of the manuscript "Structural Insights into redox signal transduction mechanisms in the control of nitrogen fixation by the NifLA system" into PNAS and the preparation of a dissertation describing the progress made on the goals of this fellowship in detail, my dissertation committee decided I was ready for my final exam. After a successful defense of his dissertation, PD Boyer was awarded a PhD in Molecular Plant Sciences, concluding the work on this project. What opportunities for training and professional development has the project provided?In executing the goals of this project, I have had the opportunity to receive training in protein purification and handling, experimental design, and project management from my mentor John Peters, as well as training from experts in X-ray diffraction and SAXS for our experiments at SSRL and the Cornell High Energy Synchrotron Source (CHESS). Additionally,k the funding from this fellowship allowed me to attend the 25th annual North American Symbiotic Nitrogen Fixation Conference in Madison, WI where I was able to share our progress and interact with biological nitrogen fixation researchers from all education levels in industry and academia. How have the results been disseminated to communities of interest?The results from this project have been demonstrated to communities of interest through the publication of a manuscript in PNAS, posts on LinkedIn, and an outreach article published in Futurum Careers, a magazine targeted at high-school students. Additionally, the results from this project were presented in public talks and poster sessions. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Determine the genetic requirements for interpretation and response to environmental signals in AvNifL. Replace nifL in the Azotobacter vinelandii chromosome with 3 ORFs encoding each of the domains of NifL to be individually transcribed and expressed. Azotobacter vinelandii strain with native nifL gene replaced via homologous recombination with 3 ORFs encoding each of the domains of NifL has been cloned, isolated, and confirmed by sequence analysis. Analyze the phenotype of modular NifL in vivo. To analyze the ability of this strain to respond to nitrogen signals, I conducted growth curves in the presence and absence of nitrogen. Growth was evaluated by measuring the optical density of culture samples collected every 3 hours throughout the course of growth. Interestingly, the modular NifL strain retained the ability to inhibit nitrogenase expression in the presence of nitrogen, demonstrating that the modular expression of NifL's domains produces a functional NifL protein. This is explained in detail in my dissertation. Monitor conditional nifH expression with qRT-PCR in the modular domain NifL strain in response to fixed nitrogen and oxygen. Generate Av strains with in-frame deletions of NifL domains using homologous recombination. Specific planned deletion strains are PAS1 and PAS2, PAS2, Q-linker, and GHKL. Analyze the phenotype of each in-frame domain deletion strain in vivo. Cell samples for RNA extraction were collected during growth curves with this strain in the presence and absence of fixed nitrogen and are ready for extraction and analysis. Although, samples were not analyzed due to ambiguity in interpreting the results and a change in strategy described in 2.2. Monitor conditional nifH expression with qRT-PCR in each domain deletion strain generated in response to fixed nitrogen and oxygen. Constructs to generate the desired domain deletions have been designed, and samples for qRT-PCR analysis will be collected from cultures of the NifL domain deletion strains throughout the course of growth in the presence and absence of fixed nitrogen or oxygen stress. Elucidate the molecular mechanism of signal perception, propagation, and response to environmental queues by AvNifL. Define an appropriate reductant for AvNifL. I performed spectroscopic analysis on purified NifL to monitor the oxidation state of its FAD cofactor which perceives the presence of oxygen. Spectra of NifL in the presence of various reductants were collected and analyzed for the presence of a peak at 450 nm, corresponding to oxidized NifL. Using this method, we identified 1,1′-bis(3-sulfonatopropyl)- 4,4′- bipyridinium(S2V), a methyl viologen derivative that allows colorimetric assessment of redox status as an appropriate reductant for NifL. Screen for crystallization conditions for the oxidized and reduced conformations of NifL in all possible nucleotideregimes and resolve the structure of each NifL conformer using X-ray diffraction. I designed and performed a stability assay based on dynamic scanning fluorimetry to optimize NifL stability and enhance our chances of crystallization. Using our qRT-PCR machine to perform thermal melts, I screened a range of buffers, pH, and osmolytes to identify optimal buffer conditions for NifL. Then, I screened NifL for crystallization conditions in all desired poised states using commercially available screens from Hampton Research. All putative NifL crystals were sent to the Stanford Synchrotron Radiation Lightsource(SSRL) for x-ray diffraction. Unfortunately, diffraction revealed no NifL crystals. Leveraging recent advances in protein structure prediction, I generated and validated a model of the NifL dimer using a combination of native mass spectrometry, dynamic light scattering (DLS) and small angle x-ray scattering (SAXS) to determine the oligomeric state and absolute dimensions of NifL in solution. Inform redox and nucleotide-induced conformational changes in NifL protein structure using mass spectrometry coupled chemical cross-linking, HDE, and surface labeling. Analyzing the structure of NifL in oxidizing or reducing conditions in the presence of ADP or ATP with surface labeling coupled mass spectrometry and SAXS-derived electron density maps reveals full-length conformational changes in the structure of NifL in response to regulatory signals. This data provides the basis for a model of signal transduction controlling nitrogenase gene expression in A. vinelandii. Optimize protocol for production and purification of deuridylylated AvGlnK. I designed and cloned E. coli strains for homologous expression of His-GlnK and GlnK(Y51F), a variant that cannot be uridylylated. Expression and purification protocols for both constructs were optimized, but we were unable to obtain sufficient GlnK(Y51F) for our experiments. Therefore, we deuridylylated His-GlnK in vivo prior to purification by ammonia shock. Screen for conditions to crystalize the NifL-GlnK complex and resolve the structure of this complex using X-ray diffraction. Since NifL is recalcitrant to crystallization, we performed anoxic SAXS at CHESS to resolve the structure of GlnK and the NifL-GlnK complex. Results from these experiments reveal an extended conformation in the GlnK T-loop in the presence of ATP and 2-oxoglutarate. However, our results suggest that the GlnK-NifL complex is unstable, and we were unable to obtain a sufficient signal to resolve the structure of the GlnK-NifL complex. Identify NifL-GlnK protein interactions and the conformation of NifL in complex with GlnK using mass spectrometry coupled chemical cross-linking, HDE, and surface labeling. GlnK-NifL samples were prepared and sent to collaborators in the Bothner lab at Montana State University. Although, we were unable to analyze these samples prior to my defense due to instrument malfunction.
Publications
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Progress 06/15/22 to 06/14/23
Outputs
Target Audience:Throughout the duration of this project, I reached the research scientist and undergraduate student subsets of our target audience through sharing the work we've conducted in this project at scientific conferences and the publication of a manuscript in PNAS. I presented a poster detailing our progress on this project at the 2022 and 2023 WSU-NIH Protein Biotechnology Training Program Symposia, which feature industry speakers and are attended primarily by WSU graduate students and professors, and I used the support of this project to attend the 25th North American Symbiotic Nitrogen Fixation Conference held in Madison, WI, where I reached industry and academic research scientists as well as graduate and undergraduate students that are working on various aspects of biological nitrogen fixation. Additionally, the work in this project was featured in a Futurum Careers article targeted at high-school-age students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In executing the goals of this project, I have had the opportunity to receive training in protein purification and handling, experimental design, and project management from my mentor John Peters, as well as training from experts in X-ray diffraction and SAXS for our experiments at SSRL and the Cornell High Energy Synchrotron Source (CHESS). Additionally,k the funding from this fellowship allowed me to attend the25th annual North American Symbiotic Nitrogen Fixation Conference in Madison, WI where I was able to share our progress and interact with biological nitrogen fixation researchers from all education levels in industry and academia. How have the results been disseminated to communities of interest?The results from this project have been demonstrated to communities of interest through the publication of a manuscript in PNAS, posts on LinkedIn, and an outreach article published in Futurum Careers, a magazine targeted at high-school students. Additionally, the results from this project were presented in public talks and poster sessions. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Determine the genetic requirements for interpretation and response to environmental signals inAvNifL. ReplacenifLin theAzotobacter vinelandiichromosome with 3 ORFs encoding each of the domains of NifL to be individually transcribed and expressed. Azotobacter vinelandii strain with native nifL gene replaced via homologous recombination with 3 ORFs encoding each of the domains of NifL has been cloned, isolated, and confirmed by sequence analysis. Analyze the phenotype of modular NifLin vivo. To analyze the ability of this strain to respond to nitrogen signals, I conducted growth curves in the presence and absence of nitrogen. Growth was evaluated by measuring the optical density of culture samples collected every 3 hours throughout the course of growth. Interestingly, the modular NifL strain retained the ability to inhibit nitrogenase expression in the presence of nitrogen, demonstrating that the modular expression of NifL's domains produces a functional NifL protein. This is explained in detail in my dissertation. Monitor conditionalnifHexpression with qRT-PCR in the modular domain NifL strain in response to fixed nitrogen and oxygen. GenerateAvstrains with in-frame deletions of NifL domains using homologous recombination. Specific planned deletion strains are PAS1 and PAS2, PAS2, Q-linker, and GHKL. Analyze the phenotype of each in-frame domain deletion strainin vivo. Cell samples for RNA extraction were collected during growth curves with this strain in the presence and absence of fixed nitrogen and are ready for extraction and analysis. Although, samples were not analyzed due to ambiguity in interpreting the results anda change in strategy described in 2.2. Monitor conditionalnifHexpression with qRT-PCR in each domain deletion strain generated in response to fixed nitrogen and oxygen. Constructs to generate the desired domain deletions have been designed, and samples for qRT-PCR analysis will be collected from cultures of the NifL domain deletion strains throughout the course of growth in the presence and absence of fixed nitrogen or oxygen stress. Elucidate the molecular mechanism of signal perception, propagation, and response to environmental queues byAvNifL. Define an appropriate reductant forAvNifL. I performed spectroscopic analysis on purified NifL to monitor the oxidation state of its FAD cofactor which perceives the presence of oxygen. Spectra of NifL in the presence of various reductants were collected and analyzed for the presence of a peak at 450 nm, corresponding to oxidized NifL. Using this method, we identified 1,1′-bis(3-sulfonatopropyl)-4,4′- bipyridinium(S2V), a methyl viologen derivative that allows colorimetric assessment of redox status as an appropriate reductant for NifL. Screen for crystallization conditions for the oxidized and reduced conformations of NifL in all possible nucleotide regimes and resolve the structure of each NifL conformer using X-ray diffraction. I designed and performed a stability assay based on dynamic scanning fluorimetry to optimize NifL stability and enhance our chances of crystallization. Using our qRT-PCR machine to perform thermal melts, I screened a range of buffers, pH, and osmolytes to identify optimal buffer conditions for NifL. Then, I screened NifL for crystallization conditions in all desired poised states using commercially available screens from Hampton Research. All putative NifL crystals were sent to the Stanford Synchrotron Radiation Lightsource(SSRL) for x-ray diffraction. Unfortunately, diffraction revealed no NifL crystals. Leveraging recent advances in protein structure prediction, I generated and validated a model of the NifL dimer using a combination of native mass spectrometry, dynamic light scattering (DLS) and small angle x-ray scattering (SAXS) to determine the oligomeric state and absolute dimensions of NifL in solution. Inform redox and nucleotide-induced conformational changes in NifL protein structure using mass spectrometry coupled chemical cross-linking, HDE, and surface labeling. Analyzing the structure of NifL in oxidizing or reducing conditions in the presence of ADP or ATP with surface labeling coupled mass spectrometry and SAXS-derived electron density maps reveals full-length conformational changes in the structure of NifL in response to regulatory signals. This data provides the basis for a model of signal transduction controlling nitrogenase gene expression in A. vinelandii. Optimize protocol for production and purification of deuridylylatedAvGlnK. I designedand cloned E. coli strains for homologous expression of His-GlnK andGlnK(Y51F), a variant that cannot be uridylylated. Expression and purification protocols for both constructs were optimized, but we were unable to obtain sufficient GlnK(Y51F) for our experiments. Therefore, we deuridylylated His-GlnK in vivo prior to purification by ammonia shock. Screen for conditions to crystalize the NifL-GlnK complex and resolve the structure of this complex using X-ray diffraction. Since NifL is recalcitrant to crystallization, we performed anoxic SAXS at CHESS to resolve the structure of GlnK and the NifL-GlnK complex. Results from these experiments reveal an extended conformation in the GlnK T-loop in the presence of ATP and 2-oxoglutarate. However, our results suggest that the GlnK-NifL complex is unstable, and we were unable to obtain a sufficient signal to resolve the structure of the GlnK-NifL complex. Identify NifL-GlnK protein interactions and the conformation of NifL in complex with GlnK using mass spectrometry coupled chemical cross-linking, HDE, and surface labeling. GlnK-NifL samples were prepared and sent to collaborators in the Bothner lab at Montana State University. Although, we were unable to analyze these samples prior to my defense due to instrument malfunction.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Nathaniel R. Boyer et al, Structural insights into redox signal transduction mechanisms in the control of nitrogen fixation by the NifLA system, Proceedings of the National Academy of Sciences (2023). DOI: 10.1073/pnas.2302732120
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
https://futurumcareers.com/Issue-21.pdf Page 92
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Progress 06/15/21 to 06/14/22
Outputs
Target Audience:In this reporting period, I primarily reached theresearch scientist and undergraduate student subsets of our target audiance through sharing the work we've conducted in this project at scientific conferences. I presentd a poster detailing our progress on this project at the 2022 WSU-NIH Protein Biotechnology Training Program Symposium, which features industry speakers and is attended primarily by WSU graduate students and professors. In June, I used the support of this project to attendthe 25th North American Symbiotic Nitrogen Fixation Conference held in Madison, WI. Here, I reached industry and academic research scientists as well as graduate and undergraduate students that are working on various aspects of biological nitrogen fixation. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In executing the goals of this project, I have had the opportunity to receive training in protein purification and handling, experimental design, and project management from my mentor John Peters, as well as training from experts in X-ray diffraction and SAXS for our experiments at SSRL. The funding from this fellowship allowed me to attend the 25th annual North American Symbiotic Nitrogen Fixation Conference in Madison, WI where I was able to share our progress and interact with biological nitrogen fixation researchers from all education levels in industry and academia. Additionally, I presented a poster detailing the progress of this project at "Biotech on and Beyond the Bench", the 2022 symposium hosted annually by the WSU-NIH Biotechnology Training Program, and I served as a poster judge for participants in the WSU Research and Extension Experiences for Undergraduates (REEU) poster competition where I was able to interact with aspiring researchers and extension agents. How have the results been disseminated to communities of interest?Results from this project have been disseminated through poster presentations and interactions with undergraduate researchers from the WSU REEU program. Currently, we are in the process of writing up the results from our studies for publication. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Access to available nitrogen commonly limits plant growth and the agricultural demand for nitrogen is largely supplemented with synthetic nitrogen fertilizers, which cause deleterious environmental effects including soil acidification and greenhouse gas emissions. Diazotrophs are bacteria and archaea that use the enzyme nitrogenase to reduce atmospheric nitrogen into ammonia that plants readily incorporate into biomolecules. Using diazotrophs in agriculture provides an avenue to reduce dependence on synthetic fertilizers, thereby improving land stewardship and agricultural sustainability. The goal of the proposed research is to understand how diazotrophs regulate nitrogenase, knowledge that is required to engineer high-performance biofertilizers from diazotrophs compatible with a wide variety of crops. During the first year of this project,I generated and validated a structural model of NifL, the sensor inhibitor protein responsible for perceiving and responding to environmental signals to modulate the expression of nitrogenase in Azotobacter vinelandii. The structural model of NifL allows us to formulate more specific questions regarding the mechanism by which NifL regulates biological nitrogen fixation as we continue with the project. More broadly, the progress in our project informs the regulation of nitrogenase in proteobacteria, a class of free-living diazotrophs that contains many of the most promising candidates biofertilizers. Determine the genetic requirements for interpretation and response to environmental signals inAvNifL. ReplacenifLin theAzotobacter vinelandiichromosome with 3 ORFs encoding each of the domains of NifL to be individually transcribed and expressed. Azotobacter vinelandiistrain with nativenifLgene replaced via homologous recombination with 3 ORFs encoding each of the domains of NifL has been cloned, isolated, and confirmed by sequence analysis. Analyze the phenotype of modular NifLin vivo. To analyze the ability of this strain to respond to nitrogen signals, I conducted growth curves in the presence and absence of nitrogen. Growth was evaluated by measuring the optical density of culture samples collected every 3 hours throughout the course of growth. Interestingly, the modular NifL strain retained the ability to inhibit nitrogenase expression in the presence of nitrogen, demonstrating that the modular expression of NifL's domains produce a functional NifL protein. Monitor conditionalnifHexpression with qRT-PCR in the modular domain NifL strain in response to fixed nitrogen and oxygen. Cell samples for RNA extraction were collected during growth curves with this strain in the presence and absence of fixed nitrogen and are ready for extraction and analysis. GenerateAvstrains with in-frame deletions of NifL domains using homologous recombination. Specific planned deletion strains are PAS1 and PAS2, PAS2, Q-linker, and GHKL. The efficacy of our genetic manipulation strategy was demonstrated by successfully creation of the modular NifL strain described in goals 1.1 and 1.2. Using the same strategy, I have designed constructs to produce the desired NifL domain deletions. Analyze the phenotype of each in-frame domain deletion strainin vivo. The initial modular NifL strain maintains the ability to respond to fixed nitrogen, demonstrates the validity of our approach. When all desired strains have been generated, we will perform similar growth curves to analyze the role of each domain in NifL signal transduction. Monitor conditionalnifHexpression with qRT-PCR in each domain deletion strain generated in response to fixed nitrogen and oxygen. Constructs to generate the desired domain deletions have been designed, and samples for qRT-PCR analysis will be collected from cultures of the NifL domain deletion strains throughout the course of growth in the presence and absence of fixed nitrogen or oxygen stress. Elucidate the molecular mechanism of signal perception, propagation, and response to environmental queues byAvNifL. Define appropriate reductant forAvNifL. I performed spectroscopic analysis on purified NifL to monitor the oxidation state of its FAD cofactor which perceives the presence of oxygen. Spectra of NifL in the presence of various reductants were collected and analyzed for the presence of a peak at 450 nm, corresponding to oxidized NifL. Using this method, we identified 1,1′-bis(3-sulfonatopropyl)-4,4′-bipyridinium(S2V), a methyl viologen derivative that allows colorimetric assessment of redox status as an appropriate reductant for NifL. Screen for crystallization conditions for the oxidized and reduced conformations of NifL in all possible nucleotide regimes and resolve the structure of each NifL conformer using X-ray diffraction. I designed and performed a stability assay based on dynamic scanning fluorimetry to optimize NifL stability and enhance our chances of crystallization. Using our qRT-PCR machine to perform thermal melts, I screened a range of buffers, pH, and osmolytes to identify optimal buffer conditions for NifL. Then, I screened NifL for crystallization conditions in all desired poised states using commercially available screens from Hampton Research. All putative NifL crystals were sent to the Stanford Synchrotron Radiation Lightsource(SSRL) for x-ray diffraction. Unfortunately, diffraction revealed no NifL crystals. Leveraging recent advances in protein structure prediction, I generate model of NifL that is consistent with expectations from the literature. To experimentally validate this model, I performed dynamic light scattering (DLS) and small angle x-ray scattering (SAXS) to determine the absolute dimensions of NifL and our collaborators from the Bothner lab at MSU confirmed the oligomeric state using native mass spectrometry. Results from both DLS and SAXS closely match the dimensions predicted by the model, and native mass spectrometry confirmed that NifL is predominantly a dimer in solution, offering independent support to validate our model of NifL. Inform redox and nucleotide regime induced conformational changes in NifL protein structure using mass spectrometry coupled chemical cross-linking, HDE, and surface labelling. I purified NifL and prepared samples with NifL poised in all oxidation states and nucleotide regimes to send to our collaborators in the Bothner lab at MSU. The Bothner lab performed chemical crosslinking and surface labeling experiments analyzed by SDS-PAGE and mass spectrometry respectively. In preliminary experiments, chemical crosslinking supports the dominant oligomeric state of NifL to be dimeric, and surface labeling illuminated differences in label incorporation between the conformations of NifL. Optimize protocol for production and purification of deuridylylatedAvGlnK. I designed a construct to express the GlnK(Y51F) variant which cannot be uridylylated and cloned this construct into E. coli for expression. Since GlnK(Y51F) cannot be uridylylated, it will constitutively interact with NifL, which is ideal for our studies of NifL-GlnK interactions. Screen for conditions to crystalize the NifL-GlnK complex and resolve the structure of this complex using X-ray diffraction. I have defined facile conditions to produce large quantities of pure, high quality NifL, and cloned the GlnK(Y51F) construct into an E. coli expression strain. Once GlnK(Y51F) purification has been optimized, I will begin performing crystallization trials with the NifL-GlnK complex. Identify NifL-GlnK protein interactions and the conformation of NifL in complex with GlnK using mass spectrometry coupled chemical cross-linking, HDE, and surface labelling. Our collaborators in the Bothner lab have optimized conditions to perform chemical cross-linking and surface labeling with NifL that will also be applicable for experiments with the NifL-GlnK complex.
Publications
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