Purification and Structural Characterization of Siderophore (Corynebactin) from Corynebacterium diphtheriae

During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin

Thermodynamics and Kinetics of Adaptive Binding in the Malachite Green RNA Aptamer

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/bi400549sAdaptive binding, the ability of molecules to fold themselves around the structure of a ligand and thereby incorporating it into their three-dimensional fold, is a key feature of most RNA aptamers. The malachite green aptamer (MGA) has been shown to bind several closely related triphenyl dyes with planar and nonplanar structures in this manner. Competitive binding studies using isothermal titration calorimetry and stopped flow kinetics have been conducted with the aim of understanding the adaptive nature of RNA–ligand interaction. The results of these studies reveal that binding of one ligand can reduce the ability of the aptamer pocket to adapt to another ligand, even if this second ligand has a significantly higher affinity to the free aptamer. A similar effect is observed in the presence of Mg2+ ions which stabilize the binding pocket in a more ligand bound-like conformation.National Science and Engineering Research Council (NSERC) [326911-2009]Canada Foundation for Innovation (CFI

Design of a motorcycle steering damper for a safer ride

Powered-two-wheelers (PTWs) are increasingly popular because of their lower cost compared to cars, and therefore the riders' exposure risk is increasing. Due to their complex dynamics characterized by high non-linearity and inherent instability, PTWs are more difficult to control compared to four-wheeled vehicles. Wobble is a high-frequency instability mode affecting the steering assembly of the PTW, and which often causes the rider to lose control and crash when it occurs. In this paper, we present the design of a new motorcycle semi-active steering damper integrated into the steering column and utilizing a magnetorheological fluid (MRF) for variable damping torque. An analytical model of the concept was first used to perform the preliminary sizing, followed by concept validation using a 3D FE multiphysics magnetic-fluid analysis. The final innovative design offers several advantages compared to traditional steering dampers: (i) a wide range of adjustable damping torque values, with a multiplication factor up to 10 with a maximum electrical current of 2 A; (ii) total integration into the motorcycle steering column enabled by its axial design and limited radius; (iii) a simple chamber geometry that allows for easy manufacture; (iv) longer seal life due to the absence of direct contact between seals and the MRF