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• 1 2020-05-06T00:05:19+00:00 Christine Lo Verde 7 plain 2020-05-08T23:50:27+00:00

  
  Structural and Biophysical Characterization of CowN from Gluconacetobacter diazotrophicus

  Overview: Soil dwelling bacteria use the enzyme nitrogenase to generate the important plant nutrient ammonia. However, the pollutant gas, carbon monoxide, prevents nitrogenase from functioning properly. Our research investigates how the bacterial protein CowN protects nitrogenase from the effects of carbon monoxide and increases bacterial ammonia production.
  
  Abstract: The bacterium Gluconacetobacter diazotrophicus expresses the enzyme nitrogenase, which converts atmospheric dinitrogen to ammonia- a critical source of nitrogen for plants. In the presence of the environmental gas-carbon monoxide (CO)-nitrogenase is inhibited. However, CowN, a protein found within many diazotrophs, can prevent CO from exerting its inhibitory effects on nitrogenase. CowN not only protects nitrogenase against CO, but is shown to have elevated expression in cold temperatures, suggesting that CowN may function as a cold response protein. Given the limited research conducted on CowN, its structure and function remain mostly unknown. Therefore, we aim to gain an understanding of the structural and biophysical properties of CowN and determine how CowN shields nitrogenase from CO. 
  
  Similar to the amyloidogenic protein Aβ, CowN aggregates as a result of changes in temperature, concentration of salt and concentration of protein. Aiming to explore how these various factors change the oligomeric state of CowN, we hypothesize that a) CowN aggregation is caused by protein secondary structural changes that are thermally induced, b) aggregation is driven by increased salt and protein concentration, and c) monomeric and oligomeric states have different activities. To test these hypotheses, CowN was purified following expression within E. coli and then functionally examined using dynamic light scattering, circular dichroism spectroscopy, and Fourier transform infrared spectroscopy. Results indicate that there are two different aggregation mechanisms occurring. At pH 7.5 and above, CowN aggregates when heated. Under these conditions, aggregation and thermal denaturation both occur around 45⁰c. Aggregation is likely caused by an amyloid-like secondary structure change from α-helices to β-sheets. At pH 7 and below, CowN aggregates at room temperature prior to unfolding. This mechanism is likely driven by electrostatics, as the pH approaches the protein’s pI. Together, data suggests that CowN is active within a narrow pH, temperature and concentration window. 
  
  
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