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Principal Investigator: Rebeccah Warmack
Organization: CALIFORNIA INSTITUTE OF TECHNOLOGY
Fiscal Year: 2024
Award: $123,446
Funding agency: National Institute of General Medical Sciences
PROJECT SUMMARY. Nitrogen fixation by the metalloenzyme nitrogenase supports nearly 50% of the global
population and is the only biological pathway for nitrogen reduction. Nitrogenase consists of two proteins, the
obligate reductase Fe-protein and the catalytic MoFe-protein, both of which are rapidly inactivated by oxygen.
Biochemical, crystallographic, and spectroscopic studies have yielded seminal insights into the structures and
functions of purified nitrogenase proteins, but a unified understanding of the enzymatic mechanism is still
unrealized. These proteins are expressed in a small subset of prokaryotes termed diazotrophs, including aerobes
and anaerobes, which inhabit a diverse array of environments. Despite this evolutionary demonstration of
compatibility between nitrogen fixation and a variety of metabolisms, robust heterologous expression of the
nitrogenase proteins has not been achieved. Instead, endogenous expression within the free-living soil bacterium
Azotobacter vinelandii remains the most widely used system for the purification and study of nitrogenase. Several
peculiar features of this obligate aerobe have been annotated under nitrogen-limited conditions, to wit, the
formation of an intracytoplasmic membrane network, but a comprehensive investigation of these features and
their relationship to the nitrogenase proteins is lacking. The primary hypothesis of this proposal is that
interactions with cellular ultrastructures and as yet unidentified binding partners in vivo significantly
regulate and promote nitrogenase activity. My goal is to identify and characterize these states of nitrogenase
in diazotrophic organisms by training in and applying emerging cryoelectron tomography (cryoET) methodologies
and performing experiments outlined in two Aims. Aim 1: Revealing the architecture of the nitrogenase
interactome in A. vinelandii with mass spectrometry and single particle (SP) cryoEM. Aim 2: Determining in situ
structures of nitrogenase complexes in conjunction with cellular features using cryogenic correlative light and
electron microscopy (cryoCLEM/ET) and sub-tomogram averaging (STA). As a postdoctoral fellow I have gained
expertise in anaerobic SP cryoEM to obtain high resolution structures of nitrogenase. These skills will provide a
foundation for the proposed research. However, I seek training in the growing field of cryoET for the study of in
situ structures. Throughout the outlined aims, I will train in genetic manipulation of non-model organisms, mass
spectrometry, and cryoET from my advisory committee, the Caltech CryoEM and proteomic facilities, and
national facilities while continuing to expand my mastery of SP cryoEM and metalloenzyme chemistry. In my
independent career, I will apply this training to the continued study of nitrogenase and other anaerobic systems
relevant to environmental and human health. The proposed aims will yield the first comprehensive investigation
of nitrogenase in situ with the increasingly high resolution technique of cryoET, and will provide insight not only
into cellular regulation of nitrogen fixation, but also structures of true turnover states within the cell thus
fundamentally moving our understanding of this important biogeochemical process forward.
Terms: <A vinelandii><A. vinelandii><Advisory Committees><Aerobic><Aerobic Bacteria><Agriculture><Ammonia><Anaerobic Bacteria><Architecture><Award><Azotobacter vinelandii><Bacteria><Binding><Biochemical><Biochemical Reaction><Biological><Catalogs><Catalytic Core><Catalytic Domain><Catalytic Region><Catalytic Site><Catalytic Subunit><Cell Body><Cell Isolation><Cell Segregation><Cell Separation><Cell Separation Technology><Cell membrane><Cells><Cellular Regulation><Chemistry><Classification><Complex><Coupled><Cryo-electron Microscopy><Cryo-electron tomography><Cryoelectron Microscopy><Crystallographies><Crystallography><Cytolysis><Cytoplasmic Membrane><Data><Dehydrogenases><Dinitrogenase><Earth><Electron Cryomicroscopy><Electron Microscopy><Engineering / Architecture><Environment><Enzymatic Reaction><Enzyme Gene><Enzymes><Fe element><FeMo Cofactor><Fellowship><Foundations><Funding><Generalized Growth><Genes><Goals><Growth><Health><Human><In Situ><In Vitro><Intermediary Metabolism><Investigation><Ions><Iron><Iron-Molybdenum Cofactor><Life><Light><Lysis><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Membrane><Metabolic><Metabolic Processes><Metabolism><Metalloproteins><Methodology><MoFe Protein><Modern Man><Molecular><Molecular Interaction><Molybdenum-Iron Protein><Molybdoferredoxin><Multienzyme Complexes><NIH><National Institutes of Health><Nif Genes><Nitrogen><Nitrogen Fixation><Nitrogenase><O element><O2 element><Organism><Oxidoreductase><Oxidoreductase Gene><Oxygen><Pathway interactions><Phase><Photoradiation><Planet Earth><Plasma Membrane><Population><Postdoc><Postdoctoral Fellow><Process><Production><Prokaryotae><Prokaryotic Cells><Proteins><Proteomics><Reductases><Research><Research Associate><Resolution><Role><Seminal><Soil><Stress><Structure><Structure-Activity Relationship><System><Systematics><Targeted Research><Task Forces><Techniques><Temperature><Tissue Growth><Training><United States National Institutes of Health><Vesicle><Wit><Work><advisory team><aerobe><anaerobe><biologic><career><catalog><cell growth regulation><cell sorting><chemical structure function><cryo-EM><cryo-EM tomography><cryoEM><cryoEM tomography><cryoelectron tomography><cryogenic electron microscopy><cryogenics><deprivation><design><designing><electron cryo-tomography><electron tomography><enzyme complex><enzyme mechanism><experiment><experimental research><experimental study><experiments><gene manipulation><genetic manipulation><genetically manipulate><genetically perturb><genome editing><genomic editing><in vivo><innovate><innovation><innovative><insight><knock-down><knockdown><light microscopy><living system><membrane structure><metalloenzyme><new technology><novel><novel technologies><ontogeny><paired helical filament><particle><pathway><plasmalemma><post-doc><post-doctoral><post-doctoral trainee><pressure><programs><prokaryote><protein complex><protein structure><protein structures><proteins structure><reconstruction><repair><repaired><research associates><resolutions><response><skills><social role><spectroscopic study><spectroscopic survey><structure function relationship><tomography>