Primer design
Interdisciplinary Scientific Research Center
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Bio STem Academy Of Research
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Bold Meaningful Scientific Innovative Research
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Protein-protein, Protein-antibody, Protein-substrate interactions
Bio STAR, LLC
Research Opportunities
The atomic structure of several protein complexes and oligomeric proteins has been established by X-ray and computational modelling studies. Protein-protein, protein-inhibitor, antibody-antigen, protein-lipid interactions are revealed in the process. There are cases, however, where the structure of the components is known, but not that of the complex. Then, the structure of the complex may be deduced by model building. Visual inspection of molecular models aided with a large body of biochemical information can be very helpful. STAR has interest in the following areas of research:
AREA OF RESEARCH 1
Complex II, also recognized as Fumarate Reductase/Succinate dehydrogenase (FRD/SDH) is uniquely tasked with a dual role in the essential energy-producing processes of a cell. Although Complex II subunits and assembly factors form part of the same enzyme complex, mutations in their respective genes lead to significantly different clinical phenotypes. Complex II plays a pivotal role in the citric acid cycle, orchestrating the oxidation of succinate to fumarate. This intricate enzymatic process involves the transfer of two electrons to membrane-soluble quinones. Members of the complex II family possess a soluble region composed of two polypeptide chains—flavoprotein and iron protein—coupled with a membrane-spanning domain of limited conservation offering a great opportunity to investigate their individual functions. Notably, the homolog of complex II, quinol-fumarate reductase (FRD), shares a comparable function, catalyzing the reduction of fumarate to succinate, specifically during anaerobic respiration. The dynamic interplay within this enzymatic system underscores the complexity of cellular respiration and energy metabolism. Current research including use of inhibitors is exploring the elusive details of cofactor insertion, involving flavin, iron-sulfur clusters, and quinones, adding an additional layer of intrigue to our understanding of these crucial processes.
AREA OF RESEARCH 2
BC1 complex: The bc1 complex, also referred to as complex III, stands as a vital player in the electron transport chain (ETC) within cellular respiration. Its primary responsibility lies in orchestrating the transfer of electrons from ubiquinol to cytochrome c, a process pivotal for establishing a proton gradient across the inner mitochondrial membrane. This proton gradient, in turn, plays an indispensable role in ATP synthesis. Comprising multiple subunits, including cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein, the bc1 complex operates through a series of redox reactions. Ubiquinone, or coenzyme Q, serves as a mobile electron carrier, facilitating the transfer of electrons to the bc1 complex from either complex I or complex II in the electron transport chain. Inhibition of the bc1 complex by substances like antimycin A can disrupt electron transport and impede ATP synthesis. This complex holds a critical position in oxidative phosphorylation, where the energy derived from electron transfer is harnessed to pump protons across the inner mitochondrial membrane, ultimately leading to ATP production. Contemporary research, often involving the use of inhibitors, aims to unravel the intricate details of cofactor insertion within the bc1 complex. This investigation encompasses domain movement between subunits introducing an additional layer of complexity to our comprehension of these fundamental cellular processes.
AREA OF RESEARCH 3
Complex II (SQR) and Complex III (bc1 complex) have been found to team up to form a Respiratory Supercomplex (A). Complex III (Ubiquinone Cyt C Oxidoreductase) from Rhodobacter Sphaeroides. includes an active site of intriguing function. Preliminary crystallization trials data of Complex III have been done and studied and need further analysis. bc1 complex is made up of three protein subunits. Double quinone occupancy at the Qo pocket—one of the active sites in bc1 complex is key to this project and available data suggest interesting questions to answer. For example, heterologous biochemical kinetic assay between Complex II from E. coli and Complex III from R. Sphaeroides are models to explain the cooperative relationship between Complex II and II (B and C). A new hybrid system has proved to be highly accurate and reproducible to show the interaction in vitro between these two complexes and recently reported in the literature.
AREA OF RESEARCH 4
MATE (multidrug and toxic compound extrusion) proteins. These integral membrane proteins mediate the export of a broad range of unrelated compounds from cells, including antibiotics and anticancer agents, thus reducing the concentration of these compounds to sub toxic levels in target cells. Xiao He et al, (Nature, 2010, 467:991-996 ) reported the X-ray structure of NorM VC determined to 3.65Å, revealing an outward-facing conformation with two portals open to the outer leaflet of the membrane and a unique topology of the predicted 12 transmembrane helices distinct from any other known MDR transporter. The report includes a cation-binding site in close proximity to residues previously deemed critical for transport. Cation binding is suggested to induce structural changes to the inward-facing conformation, which is competent to bind substrate from the inner membrane leaflet or cytoplasm. This substrate binding can presumably cause structural changes back to the outward-facing conformation, allowing export and cation binding. In spite of intensive research, it is still not clear how multidrug transporters work. The exact location for substrate binding in either outward or inward-facing conformations has not been proposed yet. To answer a lot of burning questions, I am using a combination of molecular biology, rigid body modeling, molecular dynamics, docking, fluorescent/paramagnetic reporter groups and ultimately use spin labeling with EPR spectroscopy to understand the dynamic dimension of NorM’s structure to develop a structural basis for cation and substrate recognition and translocation. Search, identification and validation of residues shaping the binding site should illuminate determinants of substrate specificity in NorM and homologues. Further structural, biochemical, biophysical and modeling studies on MATE proteins should provide a molecular description of the dynamic processes underlying substrate transport. It would also be crucial for developing modulators that bind to and inhibit the activity of MATE proteins and other multi drug transporters, thus providing a solution to the increasing problem of multidrug resistance.
AREA OF RESEARCH 5
The functional state of the cytoplasmic domain of the human chloride transporter CLC5 (CLC5-ct) via SAXS has been explored and there are intriguing questions. The C-terminus of CLC-5, contain functional domains that play a regulatory role for nucleotide interaction. The isolated CLC5-ct has been found to bind ATP and cause a large increase in thermal stability. Also, CT+ATP exist in a monomer-dimer equilibrium which was verified by using a nucleotide free wild type and a nucleotide binding mutant. SAXS helped elucidate the mechanism of CT dimerization and the role of the dimer interphase for nucleotide binding. I identified a transient and dynamic partially unfolded state by SAXS which stabilizes in the presence of binding substrates. The reconstructed low-resolution envelope of the dimer is unexpected and is currently presented in a manuscript prepared for NSMB. Efforts to identify point mutations in traditional homodimer pairs based on reported structures that would favor the dimeric state have been unsuccessful; however, additional biochemistry has demonstrated that induced compactness of ClC5-ct is a required step in conformational maturation and forward trafficking out of the ER as presented in a manuscript submitted to PNAS. However, the results of the SAXS data and new rigid body models are indicative of a dynamic unfolded state and unexpected homodimer structure for the cytoplasmic CLC5 protein. The SAX data would complement the functional studies of the effect of nucleotides on activation of the full-length chloride transporter.
AREA OF RESEARCH 6
The unprecedented public health and economic impact of the COVID-19 pandemic caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been met with an equally unprecedented scientific response. Much of this response has focused, appropriately, on the mechanisms of SARS-CoV-2 entry into host cells, and in particular the binding of the spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2), and subsequent membrane fusion. SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Our interest in this area is modelling alternative mechanisms to inhibit the action of the spike at the membrane level.