Jun-Haeng Cho

Functional evaluation of residues in the herbicide-binding site of Mycobacterium tuberculosis acetohydroxyacid synthase by site-directed mutagenesis

Mycobacterium tuberculosis acetohydroxyacid synthase (M. tuberculosis AHAS) has been proposed to bean essential target for novel herbicide- and chemical-based antibacterial agents. Therefore, here we investigated the roles of multiple conserved herbicide-binding site residues (R318, A146, Q148, M512, and V513) in M. tuberculosis AHAS through site-directed mutagenesis by characterizing the kinetic parameters and herbicide sensitivities of various point mutants. Interestingly, all mutant enzymes showed significantly altered kinetic parameters, specifically reduced affinity towards both the substrate and cofactor. Importantly, mutation of R318 led to a complete loss of AHAS activity, indicating a key role for this residue in substrate binding. Furthermore, all mutants demonstrated significant herbicide resistance against chlorimuron ethyl (CE), with several-fold higher IC50 than that of wild type AHAS. Docking analysis also indicated that binding of CE was slightly affected upon mutation of these residues. Taken together, these data suggest that the residues examined here mediate CE binding and may also be important for the catalytic activity of AHAS. This study will pave the way for future structure-function studies of CE and will also aid the development of novel anti-tuberculosis agents based on this chemical scaffold.

Development of receptor-based inhibitory RNA aptamers for anthrax toxin neutralization

Anthrax toxin excreted by Bacillus anthracis is the key causative agent of infectious anthrax disease. In the present study, we targeted the binding of PA to the ATR/TEM8 Von Willebrand factor type A (VWA) domain, which we cloned into Escherichia coli and purified to homogeneity under denaturing conditions. To develop an anthrax toxin inhibitor, we selected and identified short single strand RNA aptamers (approximately 30mer) consisting of different sequences of nucleic acids with a high binding affinity in the 100 nanomolar range against the recombinant ATR/TEM8 VWA domain using systematic evolution of ligands by exponential enrichment (SELEX). Five candidate aptamers were further characterized by several techniques including secondary structural analysis. The inhibitor efficiency (IC50) of one of the aptamers toward anthrax toxin was approximately 5μM in macrophage RAW 264.7 cells, as determined from cytotoxicity analysis by MTT assay. We believe that the candidate aptamers should be useful for blocking the binding of PA to its receptor in order to neutralize anthrax toxin.

Characterization of acetohydroxyacid synthase I from Escherichia coli K-12 and identification of its inhibitors.

The first step in branched-chain amino acid biosynthesis is catalyzed by acetohydroxyacid synthase (EC 2.2.1.6). This reaction involves decarboxylation of pyruvate followed by condensation with either an additional pyruvate molecule or with 2-oxobutyrate. The enzyme requires three cofactors, thiamine diphosphate (ThDP), a divalent ion, and flavin adenine dinucleotide (FAD). Escherichia coli contains three active isoenzymes, and acetohydroxyacid synthase I (AHAS I) large subunit is encoded by the ilvB gene. In this study, the ilvB gene from E. coli K-12 was cloned into expression vector pETDuet-1, and was expressed in E. coli BL21 (DH3). The purified protein was identified on a 12% SDS–PAGE gel as a single band with a mass of 65 kDa. The optimum temperature, buffer, and pH for E. coli K-12 AHAS I were 37 °C, potassium phosphate buffer, and 7.5. Km values for E. coli K-12 AHAS I binding to pyruvate, Mg+2, ThDP, and FAD were 4.15, 1.26, 0.2 mM, and 0.61 μM respectively. Inhibition of purified AHAS I protein was determined with herbicides and new compounds.

Use of Multiple Peptide-Based SERS Probes Binding to Different Epitopes on a Protein Biomarker To Improve Detection Sensitivity

We propose an analytical strategy to improve the sensitivity for detecting a protein biomarker through signal multiplication by manipulating multiple peptide-based surface-enhanced Raman scattering (SERS) probes to bind the biomarker. Protective antigen (PA) was used as an Anthrax biomarker in this study. For this purpose, five small peptides selective to various PA epitopes with different binding affinities were chosen and peptide-conjugated Au nanoparticle (AuNP) SERS probes were individually prepared using each peptide. Initially, five different SERS probes were separately used to detect PA and the sensitivities were compared. Next, the possibility of enhancing sensitivity by employing multiple SERS probes was examined. Rather than applying the probes simultaneously, which would induce competitive binding, each probe was added sequentially and an optimal probe-addition sequence was determined to provide maximal sensitivity. Finally, PA samples at seven different concentrations were measured with the optimal sequence. The limit of detection (LOD) was 0.1 aM, and the enhancement was more effective at lower PA concentrations. The proposed scheme can be further applicable to detect other protein biomarkers to diagnose various diseases.

Advances in Anthrax Detection: Overview of Bioprobes and Biosensors.

Anthrax is an infectious disease caused by Bacillus anthracis. Although anthrax commonly affects domestic and wild animals, it causes a rare but lethal infection in humans. A variety of techniques have been introduced and evaluated to detect anthrax using cultures, polymerase chain reaction, and immunoassays to address the potential threat of anthrax being used as a bioweapon. The high-potential harm of anthrax in bioterrorism requires sensitive and specific detection systems that are rapid, field-ready, and real-time monitoring. Here, we provide a systematic overview of anthrax detection probes with their potential applications in various ultra-sensitive diagnostic systems.

A novel peptide-based recognition probe for the sensitive detection of CD44 on breast cancer stem cells

Metastasis and recurrence of breast cancer remain significant clinical problems. The expression level of CD44 protein is higher in breast cancer-initiating cancer stem cells; therefore, the early detection of CD44 using a sensitive diagnostic probe is important for breast cancer diagnosis and therapeutic purposes. In this study, we fabricated a polyvalent directed peptide polymer (PDPP) that specifically recognized the CD44 biomarker, as confirmed by immunocytochemistry tests and fluorescence-activated cell sorting assessment. Our results indicate that PDPP is useful as a novel tool for the sensitive detection of breast cancer stem cells.

Feasibility of asymmetrical flow field-flow fractionation as a method for detecting protective antigen by direct recognition of size-increased target-captured nanoprobes.

Asymmetrical flow field-flow fractionation (AF4) was evaluated as a potential analytical method for detection of a protective antigen (PA), an Anthrax biomarker. The scheme was based on the recognition of altered AF4 retention through the generation of the size-increased Au nanoparticle probes as a result of PA binding, in which a PA-selective peptide was conjugated on the probe surface. In the visible absorption-based AF4 fractograms, the band position shifted to a longer retention time as the PA concentration increased due to the presence of probe bound with PAs. The shift was insignificant when the concentration was relatively low at 84.3pM. To improve sensitivity, two separate probes conjugated with two different peptides able to bind on different PA epitopes were used together. The band shift then became distinguishable even at 84.3pM of PA sample. The formation of larger PA-probe inter-connected species using the dual-probe system was responsible for the enhanced band shift. In parallel, the feasibility of surface-enhanced Raman scattering (SERS) as a potential AF4 detection method was also evaluated. In the off-line SERS fractogram constructed using fractions collected during AF4 separation, a band shift was also observed for the 84.3pM PA sample, and the band intensity was higher when using the dual-probe system. The combination of AF4 and SERS is promising for the detection of PA and will become a potential tool if the reproducibility of SERS measurement is improved.

Optical Sensing Properties of ZnO Nanoparticles Prepared by Spray Pyrolysis

We studied the optical sensing properties of ZnO nanoparticles prepared by spray pyrolysis. To investigate their optical sensing performance, we incubated peptides on ZnO nanoparticles. The photoluminescence (PL) peak intensity of peptides on the ZnO nanoparticles was higher than that of peptides on the ZnO film or on the glass plate. This observed PL enhancement is attributed to the optical confinement of ZnO nanoparticles. The low-temperature spectra displayed a strong exciton emission peak with multiple sidebands, attributed to the bound exciton and its longitudinal optical phonon sidebands. The strong exciton emission is thought to be the combined effect of optical confinement due to the nanoparticle geometry, reduction of defect emission by thermal annealing, and reduction of non-radiative relaxation at low temperatures.