Eda Koculi

Selective nano-sensing approach for the determination of inorganic phosphate in human urine samples

A novel sensing approach is presented for the analysis of phosphate ions in human urine samples. The new sensor is based on the fluorescence energy transfer between a luminescent probe ([Tb-EDTA]-1) and gold nanoparticles capped with a cetyltrimethylammonium bromide (Au NPs-CTAB). The strong affinity between CTAB receptors and phosphate ions results in a highly selective assay with minimum sample preparation steps. Possible chemical interference is monitored on real-time basis via luminescence lifetime analysis. The simplicity of analysis and the competitive limit of detection in the micro-molar concentration range provide a well-suited approach for routine monitoring of phosphate ions in numerous urine samples.

DbpA is a region-specific RNA helicase.

DbpA is a DEAD‐box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA‐like catalytic core responsible for double‐helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic cores grasp. In other words, it is not known if DbpA is a site‐specific enzyme or region‐specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region‐specific or a site‐specific enzyme. Our data suggest that DbpA is a region‐specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C‐terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double‐helices in its vicinity. The only requirement for a double‐helix to serve as a DbpA substrate is for the double‐helix to be positioned within the catalytic cores grasp.

Kinetics and Thermodynamics of DbpA Protein’s C-Terminal Domain Interaction with RNA

DbpA is an Escherichia coli DEAD-box RNA helicase implicated in RNA structural isomerization in the peptide bond formation site. In addition to the RecA-like catalytic core conserved in all of the members of DEAD-box family, DbpA contains a structured C-terminal domain, which is responsible for anchoring DbpA to hairpin 92 of 23S ribosomal RNA during the ribosome assembly process. Here, surface plasmon resonance was used to determine the equilibrium dissociation constant and the microscopic rate constants of the DbpA C-terminal domain association and dissociation to a fragment of 23S ribosomal RNA containing hairpin 92. Our results show that the DbpA protein’s residence time on the RNA is 10 times longer than the time DbpA requires to hydrolyze one ATP. Thus, our data suggest that once bound to the intermediate ribosomal particles via its RNA-binding domain, DbpA could unwind a number of double-helix substrates before its dissociation from the ribosomal particles.

Time course of large ribosomal subunit assembly in E. coli cells overexpressing a helicase inactive DbpA protein

DbpA is a DEAD-box RNA helicase implicated in Escherichia coli large ribosomal subunit assembly. Previous studies have shown that when the ATPase and helicase inactive DbpA construct, R331A, is expressed in E. coli cells, a large ribosomal subunit intermediate accumulates. The large subunit intermediate migrates as a 45S particle in a sucrose gradient. Here, using a number of structural and fluorescent assays, we investigate the ribosome profiles of cells lacking wild-type DbpA and overexpressing the R331A DbpA construct. Our data show that in addition to the 45S particle previously described, 27S and 35S particles are also present in the ribosome profiles of cells overexpressing R331A DbpA. The 27S, 35S, and 45S independently convert to the 50S subunit, suggesting that ribosome assembly in the presence of R331A and the absence of wild-type DbpA occurs via multiple pathways.

Deciphering the Action Mechanism of DDX3: An RNA Helicase Implicated in Cancer Propagation and Pathogenic Viral Infection

DDX3 is a human DEAD-box RNA helicase implicated in crucial cellular processes including translation initiation, ribosome assembly, RNA transport, and microRNA processing. Consequently, DDX3 is implicated in many viral infections and cancer cell metabolism. Our goal is to obtain a detailed understanding of DDX3s mechanism of action and employ this understanding to discover DDX3 inhibitors that would serve as lead compounds for drugs that halt viral infections and cancer cell metabolism. While DDX3 is required for many viral infections and cancer cell propagations, it is not essential for healthy cell metabolism, making DDX3 an ideal anticancer and antiviral drug target. Like all the members of the DEAD-box family of enzymes, DDX3 uses ATP binding and hydrolysis to unwind short double-stranded RNA helices. Our data show that different from many members of DEAD-box family of enzymes, monomeric DDX3 is unable to perform RNA unwinding, and a multimeric DDX3 complex is required to support DDX3s helicase activity. Furthermore, our data suggests that the single-stranded-double-stranded RNA junction promotes the formation of the DDX3 multimer. We are in the process of performing mutagenesis studies combined with cross-linking and mass spectrometry to determine the DDX3 amino acids implicated in mulitmer formation and the amino acids that come in direct contact with RNA during the DDX3 catalytic cycle. These experiments would produce information both on DDX3s mechanism of action, and elucidate why some DEAD-box proteins have evolved to act as mulitmers. Lastly, we have found four natural compounds that are specific inhibitors of DDX3 ATPase activity. These compounds will be used as probes to decipher DDX3s action mechanism and could have translational potential as drugs that stop various viral infections and cancer progression.