OVERVIEW
Banerjee lab at ECU investigates the role of transition metal ions, especially copper and iron in biology. In addition, we also investigate how STEM/chemistry culture is imagined and practiced and the influence of power and politics in STEM culture and epistemology. Banerjee has a decade long experience in iron biochemistry and have mentored graduate and undergraduate students as a PI, postdoctoral fellow, and graduate student. The main techniques used in our lab are ITC (isothermal titration calorimetry), DSC (differential scanning calorimetry), UV-Vis (spectroscopy), NMR, ICP-MS (inductively coupled plasma mass spectrometry), CV (cyclic voltammetry), CD (circular dichroism), etc. In addition to getting trained on these instruments, students in Banerjee lab learn to conduct research in teams, developing peer-mentoring qualities; writing and presenting scientific work in the discipline, on other valuable soft skills. The section below will describe the three main research projects that students in Banerjee lab are currently engaged in.
OXIDATION-DEPENDENT FERROUS IRON TRANSPORTER FROM BRUCELLA SPP.
Background
Due to its high abundance on earth's crust, iron has been used as a metal co-factor, carrying essential life processes, since the start of evolution. However, the hydrophobic nature of cell membrane creates a barrier towards transport of aqueous Fe(III) across the membrane. In addition, Fe(III), the predominant oxidation state of iron under oxyc conditions, is extremly insoluble in toxic. To overcome these barriers, biological systems uptake enviroenmental iron using specilized membrane permeaes and associated proteins. One such novel membrane transporter, FtrC, is found in several human pathogens and homolgs of FtrC are also present in eukaryotes. Despite its wide presence in biology, the mechanism of iron transport through FtrC and its bacterial and eukaryotic homolgs remain a mystery. In our lab using recombinant protein method and biophysical technique we are characterizing the mechanism (thermodynamics and kinetics) of iron transport through FtrC. FtrC is co-expressed with three more protein components, periplasmic FtrA and FtrB, and inner-mmebrane bound FtrD. The accepted functional model (Ahmed el al 2013, Rajasekaran et al 2009) predict that the periplasmic FtrB as a novel cupredoxin which binds Cu(II) in an unknown and non-Type-I (HHC) copper-site and oxidizes its substrate Fe(II) to Fe(III) during its transport through FtrC. This oxidation dependent iron translocation through FtrC (and its homologs) is a crucial requirement for this family of iron transporters. Although this oxidation coupled transport has been experimentally established in the yeast Ftr system (Ftr1p-Fet3p), this remains to be established in any other eukaryotic or bacterial Ftr system.
Our wok so far
Students in Banerjee lab (Yasemene, Jacob, and Mina) in collaboration with Dr. Martin in the Brody School of Medicine have cloned and purified wild-type FtrA and four of its mutants (these mutants are predicted to show metal binding defect). Their study by Banerjee and Chanakira et al, 2020) showed that FtrA bins a Fe(II) mimic, Mn(II), in a Cu(II) dependent fashion, and mutants lacking the Cu(II) binding residues cannot bind Mn(II) or produce the functional and native fold of FtrA.
Using bioinformatics, Banerjee, Kerkan, and collaborators (2022) showed that ftrABCD is abundant in many other bacterial genomes, and based on the absence/presence of the electron sink, ftrD, two different evolution of ftrB genes was observed. This indicates some unknown yet important role that FtrD plays in the function of the FtrABCD system. Knowing that FtrD is the predicted electron sink in the FtrABCD system, the absence of this protein from the ftrABCD operon directing a different evolution for the putative Fe(II) oxidizing protein, FtrB, makes sense. However, this bioinformatics finding still needs to be experimentally verified. This work also showed that FtrC is most closely related to the only characterized Ftr system from yeat (Ftr1p) and is expected to function in a similar way, compared to other bacterial Ftr homologs. This observation is another indirect evidence that like Ftr1p, FtrC can only transport iron is Fe(II) oxidation by a protein partner is coupled with this transport.
In another work (under review), Kerkan, Banerjee, et al showed that FtrB can bind Cu(II) with a weak affinity (5-times weaker than FtrA). Based on this observation, a thermochemical cycle was constructed to determine the free energy change for the reaction where FtrB acts as Cu(II) chaperone for FtrA. This calculation yielded a negative free energy change, indicating a thermodynamically spontaneous process. Indirect evidence of FtrA-FtrB interaction mediated by Cu(II) was obtained by performing CV experiments, which indicated a complete alteration of the Cu(II) environment in the protein-protein mixture.
Currently, members of Banerjee lab are investigating this proposed FtrA-FtrB interaction and the kinetics and thermodynamics of metal oxidation/binding/release from FtrA and FtrB.
Exploiting iron(III)-siderophore transporting permeases as a drug entry point in a Trojan-Horse approach
As stated already, iron is an essential micronutrient for nearly all living organisms, including human pathogens. In a host-pathogen interface, the concentration of iron is highly restricted by the mammalian innate immune response, starving the invading pathogens. Pathogens respond to this by sending out small organic Fe(III) chelating ionophores (siderophores) to the host system to steal host protein-bound iron and these Fe(III)-siderophore complexes are actively transported into the bacterial cells using membrane transporters. Often, the ability of a bacteria to survive inside the host body depends on the expression of these Fe(III)-siderophore transporters, making these attractive drug entry points. In our lab, we are currently working on creating a siderophore-antibiotic library to test them against pathogens and determine their efficacy.
STEM culture-politics-power
STEM is in itself a society with its own etiquette, language, and rituals. In this project, we are investigating how the standardized STEM culture and language are influenced by historical events, such as European colonization, the two wars in the 1900s, sexism, racism, etc. This project is mostly student-led and investigates how this status quo and silence can be broken creating space for new voices and alternative and sustainable STEM culture. Currently, students are working on two active projects based on observation from ECU STEM undergraduate student experience perspectives. To know more about these projects, please get in touch with Banerjee.