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About the Program
About the Program
The Undergraduate Research Opportunities Program (UROP) allows undergraduate students to identify a mentor and work on a research project with them to expand the scientific knowledge and apply theory into practice.
About the Basu Lab
About the Basu Lab
The Basu Lab is an interdisciplinary research group of IUI's Chemistry and Chemical Biology department that ranges in organic chemistry, inorganic chemistry, and biochemistry. The team is particularly interested in molybdenum enzymes, which are significant in the metabolism of nitrogen, arsenic, and sulfur, and has important implications in human health and environment.
My Research Project
My Research Project
Investigation of the influence of inter-subunit interaction among E. coli NapA and NapB on the catalytic efficiency of NapAB complex
Investigation of the influence of inter-subunit interaction among E. coli NapA and NapB on the catalytic efficiency of NapAB complex
Abstract
Abstract
Periplasmic nitrate reductase (Nap) is a nitrate-reducing enzyme that is prevalent in pseudomonadota (previously known as proteobacteria). Nap is suspected to play a role in the survival of pseudomonadota, yet the formation, function, and mechanistic pathways of Nap enzymes in certain species of pseudomonadota, such as E. coli, are relatively uncharacterized. Determining the dissociation constants (KD) of the E. coli Nap enzyme complex and the rate of nitrate reduction in different molecular environments like pH levels and salt concentrations can give an insight into how environmental factors affect the survival of these bacteria. To collect this data, NapA and NapB, which are subunits of the Nap enzyme complex, will be purified. The project is still in the stages of isolating and purifying the proteins. However, it is hypothesized that the varying pH levels and salt concentrations will affect the rate of enzyme activity and the data collected can be interpreted to determine the influence of environmental factors on the enzyme complex. This information can be used to further understand the formation of the Nap enzyme complex and the mechanistic pathway of nitrate reduction via the Nap enzymes in pseudomonadota.
Background/Introduction
Background/Introduction
The molybdopterin family of enzymes is commonly found in microbes and plays a major role in the metabolization of carbon, nitrogen, arsenic, selenium, etc.1 Periplasmic nitrate reductase (Nap) is an enzyme under the molybdopterin family and reduces nitrate to nitrite (NO3 – + 2e– + 2H+ = NO2 – + H2O). The catalytic subunit of the Escherichia coli (E. coli) Nap, NapA, contains a Molybdenum cofactor (Moco) and a [4Fe4S] cluster (iron-sulfur cluster). NapB, delivers the electrons required for the reduction to NapA, and together, they form the NapAB complex in the Nap enzyme2. How the subunits interact have shown to affect the rate of the redox reaction5. Particularly, the absence of NapB from the complex affects the efficiency of the redox reaction and the binding of the substrate. NapA has also been found to have an optimal ionic and pH environment for efficient performance3. The likeliness of formation and the stability of the NapAB complex can be described by the dissociation constant (Kd). Previously, it was determined that the Kd for the NapAB complex changed during oxidation and reduction, with a value of 15 µM during oxidation and 32 µM during reduction5. A question that arises from these findings is what other factors affect the value of the dissociation constant. Prior investigations found that salt concentration and pH affect the formation of protein complexes and their stability7,8. Therefore, this research project explored how salt concentration and pH affected the interactions of the NapA and NapB subunits and gave an insight into the factors that affect the rate of nitrate reduction in E. coli Nap enzymes.
Methods
Methods
Growth and Purification
Growth and Purification
E. coli napAB with individual strep-tag in the C-terminus was obtained from GenScript and transformed into E. coli EPI400 cells. There were no previously reported standardized method of growing and purifying the E. coli cells, so variations to a previously known growth method of Campylobacter jejuni (C. jejuni) and empirical testing of the purification process was conducted to establish a standard for E. coli NapAB. The transformed E. coli cells were grown in LB media containing kanamycin and chloramphenicol overnight at 37 ºC. 10 mL of this culture was added to 1 L of autoinduction media, and the cells were grown for one day at 37 ºC and then harvested. The harvested cells were lysed and centrifuged. The protein of interest was purified from the cell lysate by affinity (StrepTrap) and size exclusion chromatography.
The E. coli NapAB was grown in an autoinduction medium with variations in additive, growth temperature, and growth length. In the first trial, the cells were grown in four flasks, with a total volume of 5 L. An additive called arabinose, which is a sugar that aids cell growth, was added to three out of the four flasks. All four flasks were grown at 37ºC for approximately 24 hours. Modifications to the second trial was made based on results from the first trial. In the second trial, the cells were grown in four flasks, with a total volume of 5 L, and no arabinose were added. Each flask had a varied growth temperature and collection date. The first flask was cultured at room temperature (approximately 24ºC) and collected in 24 hours. The second flask was cultured at 37ºC and collected in 24 hours. The third flask was cultured at room temperature and collected in 48 hours. The last flask was cultured at 37ºC and collected in 48 hours. After harvesting, the
The collected E. coli NapAB from the first trial was purified using a StrepTrap column. Multiple concentrations of biotin was utilized to remove impurities from the sample (eg. 0.05 mM, 0.1 mM, 0.2 mM) and collect the NapAB protein (eg. 20 mM). An SDS-PAGE using a 14% acrylamide gel at 120 V was performed to verify the purity of the protein. Next, a size exclusion chromatography was performed on the same sample to further purify the sample. The same SDS-PAGE procedure was also performed after the size exclusion. The protein collected from the second trial was purified and tested in a similar manner.
Kinetics - Salt Concentration and pH Level
Kinetics - Salt Concentration and pH Level
TBA
Results and Discussion
Results and Discussion
Growth and Purification
Growth and Purification
Measuring the protein concentration from the cells grown using a Bradford assay, determined that the addition of arabinose did not result in significantly more yield of E. coli cells during growth. This result allowed arabinose to be eliminated from the growth process. The protein concentration for the different temperatures and growth durations has not yet been measured.
The SDS-PAGE gels from the StrepTrap column purification showed that an impurity still remained in the sample (Figure 3). A size exclusion chromatography was performed on the same sample in the FPLC to attempt to remove the impurity. The chromatogram showed three distinct peaks (Figure 4). A griess assay was performed on the proteins in peak 1 and 2 to determine their activity and found that only the proteins in peak 2 were active in nitrate reduction. A NanoDrop UV-Vis Spectrophotometer was used to measure the spectrum of proteins in peak 3 and found a peak at 408 nm, which is consistent with the presence of heme in NapB proteins.
Figure 3. SDS-PAGE gel for E. coli NapAB (2) after StrepTrap column purification
Figure 4. FPLC size exclusion chromatogram for E. coli NapAB (0,1,2)
Conclusion
Conclusion
Conclusions TBA
Acknowledgments
Acknowledgments
Basu Lab Group Members:
Dr. Partha Basu (Principle Investigator)
Dr. Nitai Giri (Mentor)
Mikayla Metzger
Thaddaeus Broussard
Alexia Adams
Steven Benson
Mark Kibirige
Adetutu Aderinwale
References
References
1. Breeanna Mintmier; McGarry, J. M.; Bain, D. J.; Basu, P. Kinetic Consequences of the Endogenous Ligand to Molybdenum in the DMSO Reductase Family: A Case Study with Periplasmic Nitrate Reductase. Journal of Biological Inorganic Chemistry 2020, 26 (1), 13–28. https://doi.org/10.1007/s00775-020-01833-9.
2. Arnoux, P.; Sabaty, M.; Alric, J.; Frangioni, B.; Guigliarelli, B.; Adriano, J.-M.; Pignol, D.Structural and Redox Plasticity in the Heterodimeric Periplasmic Nitrate Reductase. Nature Structural & Molecular Biology 2003, 10 (11), 928–934. https://doi.org/10.1038/nsb994.
3. Sparacino-Watkins, C.; Stolz, J. F.; Basu, P. Nitrate and Periplasmic Nitrate Reductases. Chemical Society reviews 2014, 43 (2), 676–706. https://doi.org/10.1039/c3cs60249d.
5. Jepson, B.; Mohan, S.; Clarke, T. A.; Gates, A. J.; Cole, J. A.; Butler, C. S.; Butt, J. N.; Hemmings, A. M.; Richardson, D. J. Spectropotentiometric and Structural Analysis of the Periplasmic Nitrate Reductase from Escherichia Coli. Journal of Biological Chemistry 2007, 282 (9), 6425–6437. https://doi.org/10.1074/jbc.m607353200.
7. Ahl, I.-M.; Jonsson, B.-H.; Tibell, L. A. E. Thermodynamic Characterization of the Interaction between the C-Terminal Domain of Extracellular Superoxide Dismutase and Heparin by Isothermal Titration Calorimetry. Biochemistry 2009, 48 (41), 9932–9940. https://doi.org/10.1021/bi900981k.
8. Yao, X.; Chen, C.; Wang, Y.; Dong, S.; Liu, Y.-J.; Li, Y.; Cui, Z.; Gong, W.; Perrett, S.; Yao, L.; Lamed, R.; Bayer, E. A.; Cui, Q.; Feng, Y. Discovery and Mechanism of a PH-Dependent Dual-Binding-Site Switch in the Interaction of a Pair of Protein Modules. Science Advances 2020, 6 (43), eabd7182. https://doi.org/10.1126/sciadv.abd7182.
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