Biochemistry and Molecular Biology
Biophysical Characterization of CowN from Gluconacetobacter Diazotrophicus
Presenter(s): Kevin Bretzing
Advisor(s): Dr. Cedric Owens, Michael Medina
Gluconacetobacter diazotrophicus is a nitrogen fixing bacterium that is associated with plants and plays a crucial part in providing fixed nitrogen to many crops such as sugar cane. The enzyme responsible for reducing atmospheric nitrogen to ammonia is nitrogenase. The presence of carbon monoxide gas will inhibit nitrogen fixation by nitrogenase. While nitrogenase in vitro is inhibited, diazotrophs may have a mechanism of protecting nitrogenase in vivo. It is believed that a protein contained in diazotrophs, CowN, protects the nitrogenase from the detrimental effects of carbon monoxide. The overall goal of this research is to better understand how CowN shields the nitrogenase from carbon monoxide. Particularly, we are interested in understanding the structure and biophysical properties of CowN and whether it interacts directly with nitrogenase. Here, we will describe the purification of CowN and its biophysical characterization. Following expression of the CowN gene in E. coli and purification by affinity and size exclusion chromatography, samples of CowN can be found in two different states; monomeric and oligomeric. The functional importance of the two oligomeric states and the interconversion mechanism is unknown. CowN contains a cysteine residue which can form disulfide bridges with other cysteine residues. We determined that reducing the disulfide results in greater amounts of monomer, suggesting that disulfide bond formation and oligomerization may be related. Furthermore, we found that a CowN variant that has its cysteine mutated to a serine also predominantly forms a monomer. However, oligomer still is present, suggesting oligomerization is not entirely dependent on disulfide bond oxidation. Therefore, we are exploring other factors that may cause the change in oligomeric state of CowN such as temperature, salt concentration and pH. We characterize the change in oligomeric state by using dynamic light scattering, size exclusion chromatography and circular dichroism spectroscopy. The results of these experiments will be discussed in the presentation.
Determining the Structure of CowN Through Protein Crystallography
Presenter(s): Estevan Harris
Advisor(s): Dr. Cedric Owens, Michael Medina
Nitrogen fixation is the conversion of dinitrogen gas to ammonia. This process is catalyzed in nature by diazotrophic bacteria, which contain the enzyme nitrogenase. Nitrogenase uses the biological energy carrier ATP and biological reducing equivalents to reduce N2 to NH3. Biological nitrogen fixation, however, can be inhibited if there is a high concentration of carbon monoxide present. Carbon monoxide (CO) is a plant signaling gas, can also be found in high concentrations in the soil, and arises from anthropogenic sources. The protein CowN allows the survival of nitrogen fixing bacteria grown in the presence of CO. Since the mechanism and structure for CowN are not known, our goal is to characterize CowN by solving its protein crystal structure using protein crystallography. CowN is expressed recombinantly in E. coli and purified by affinity and gel filtration chromatography. Next the purity of CowN is checked by SDS PAGE. Purified protein is used set up 24-well and 96-well crystal trays. So far, we have been able to purify the CowN protein in good yield, but we have not found a suitable condition for crystal growth. Crystallography experiments are therefore ongoing.
The Effects of Gibberellic Acid and Auxin Hormones on Heliotropism in Sunflowers
Presenter(s): Brandon Bernardo
Advisor(s): Dr. Hagop Atamian
Sunflowers are one of many different plant species that are able to track and face the sun in order to optimize the amount of sunlight they are exposed to. This process of orienting towards the sun is called Heliotropism. Sunflowers are able to effectively orient themselves towards the sun because the growth rate on the East and West side of the stem alternates depending on the time of day. At dawn, the East facing stem will grow at a faster rate than the West facing side, resulting in the flower orienting towards the West. This alternating and uneven growth is what allows the sunflower to track the sun during the day and reorient at night to face the East in preparation for sunrise. Not much is known about the biological processes that induce heliotropism. In our study, we focused on two known growth inducing hormones in plants that are present in sunflowers, Gibberellic Acid and Auxin, and their importance to heliotropism. Because of their prevalence in sunflowers and their known ability to induce growth in plants, we hypothesized that Gibberellic Acid (GA) and an Auxin hormone, Indole-3-Acetic Acid (IAA), play a significant role in sunflower’s ability to perform heliotropism.