Biophysical Features of Bacillithiol, the Glutathione Surrogate of Bacillus subtilis and other Firmicutes

Bacillithiol (BSH) is the major low-molecular-weight (LMW) thiol in many low-G+C Gram-positive bacteria (Firmicutes). Evidence now emerging suggests that BSH functions as an important LMW thiol in redox regulation and xenobiotic detoxification, analogous to what is already known for glutathione and mycothiol in other microorganisms. The biophysical properties and cellular concentrations of such LMW thiols are important determinants of their biochemical efficiency both as biochemical nucleophiles and as redox buffers. Here, BSH has been characterised and compared with other LMW thiols in terms of its thiol pKa, redox potential and thiol–disulfide exchange reactivity. Both the thiol pKa and the standard thiol redox potential of BSH are shown to be significantly lower than those of glutathione whereas the reactivities of the two compounds in thiol–disulfide reactions are comparable. The cellular concentration of BSH in Bacillus subtilis varied over different growth phases and reached up to 5 mM, which is significantly greater than previously observed from single measurements taken during mid-exponential growth. These results demonstrate that the biophysical characteristics of BSH are distinctively different from those of GSH and that its cellular concentrations can reach levels much higher than previously reported

Readout fidelity of coaxial holographic digital data page recording in nanoparticle–(thiol–ene) polymer composites

We report on an experimental investigation of nanoparticle-concentration and thiol-to-ene stoichiometric ratio dependences of symbol error rates (SERs) and signal-to-noise ratios (SNRs) of digital data pages recorded at a wavelength of 532 nm in thiol–ene based nanoparticle–polymer composite (NPC) films by using a coaxial holographic digital data storage method. We show that SERs and SNRs at the optimized material condition can be lower than 1 × 10−4 and higher than 10, respectively, without error correction coding. These results show the usefulness of thiol–ene based NPCs as coaxial holographic data storage media

Enzymatic Cross-Linking of Dynamic Thiol-Norbornene Click Hydrogels

Enzyme-mediated in situ forming hydrogels are attractive for many biomedical applications because gelation afforded by enzymatic reactions can be readily controlled not only by tuning macromer compositions, but also by adjusting enzyme kinetics. For example, horseradish peroxidase (HRP) has been used extensively for in situ cross-linking of macromers containing hydroxyl-phenol groups. The use of HRP to initiate thiol-allylether polymerization has also been reported, yet no prior study has demonstrated enzymatic initiation of thiol-norbornene gelation. In this study, we discovered that HRP can generate the thiyl radicals needed for initiating thiol-norbornene hydrogelation, which has only been demonstrated previously using photopolymerization. Enzymatic thiol-norbornene gelation not only overcomes light attenuation issue commonly observed in photopolymerized hydrogels, but also preserves modularity of the cross-linking. In particular, we prepared modular hydrogels from two sets of norbornene-modified macromers, 8-arm poly(ethylene glycol)-norbornene (PEG8NB) and gelatin-norbornene (GelNB). Bis-cysteine-containing peptides or PEG-tetra-thiol (PEG4SH) was used as a cross-linker for forming enzymatically and orthogonally polymerized hydrogel. For HRP-initiated PEG-peptide hydrogel cross-linking, gelation efficiency was significantly improved via adding tyrosine residues on the peptide cross-linkers. Interestingly, these additional tyrosine residues did not form permanent dityrosine cross-links following HRP-induced gelation. As a result, they remained available for tyrosinase-mediated secondary cross-linking, which dynamically increased hydrogel stiffness. In addition to material characterizations, we also found that both PEG- and gelatin-based hydrogels exhibited excellent cytocompatibility for dynamic 3D cell culture. The enzymatic thiol-norbornene gelation scheme presented here offers a new cross-linking mechanism for preparing modularly and dynamically cross-linked hydrogels

Glutathione S-Transferase activity and total thiol status in chronic alcohol abusers before and 30 days after alcohol abstinence

Background: Glutathione S Transferase (GST) has been involved in detoxification process in the liver and its activity has been shown to be increased in alcohol abusers. In the current work we measured the GST activity, total thiol status, AST, ALT, and direct bilirubin in chronic alcohol abusers before and 30 days after alcohol abstinence and lifestyle modification. Methods: Serum and urine GST activity and total thiol status were determined using spectrophotometric methods and serum transaminases were determined using clinical chemistry analyzer. Results: We found,significant increase in serum and urine GST (p<0.001), AST (p<0.001), ALT (p<0.001), and decrease in total thiol status (p<0.001) in chronic alcohol abusers. GST activity significantly decreased (p<0.001) and total thiol status were improved significantly (p<0.001) 30 days after alcohol abstinence and lifestyle modification. Conclusion: This study provides preliminary data to suggest the role of GST as prognostic indicator of alcohol abstinence with possible trend towards an improvement in liver function

Thiol density dependent classical potential for methyl-thiol on a Au(111) surface

A new classical potential for methyl-thiol on a Au(111) surface has been developed using density functional theory electronic structure calculations. Energy surfaces between methyl-thiol and a gold surface were investigated in terms of symmetry sites and thiol density. Geometrical optimization was employed over all the configurations while minimum energy and thiol height were determined. Finally, a new interatomic potential has been generated as a function of thiol density, and applications to coarse-grained simulations are presented

Structure and function of the bacterial heterodimeric ABC transporter CydDC: stimulation of ATPase activity by thiol and heme compounds.

In Escherichia coli, the biogenesis of both cytochrome bd-type quinol oxidases and periplasmic cytochromes requires the ATP-binding cassette-type cysteine/GSH transporter, CydDC. Recombinant CydDC was purified as a heterodimer and found to be an active ATPase both in soluble form with detergent and when reconstituted into a lipid environment. Two-dimensional crystals of CydDC were analyzed by electron cryomicroscopy, and the protein was shown to be made up of two non-identical domains corresponding to the putative CydD and CydC subunits, with dimensions characteristic of other ATP-binding cassette transporters. CydDC binds heme b. Detergent-solubilized CydDC appears to adopt at least two structural states, each associated with a characteristic level of bound heme. The purified protein in detergent showed a weak basal ATPase activity (approximately 100 nmol Pi/min/mg) that was stimulated ∼3-fold by various thiol compounds, suggesting that CydDC could act as a thiol transporter. The presence of heme (either intrinsic or added in the form of hemin) led to a further enhancement of thiol-stimulated ATPase activity, although a large excess of heme inhibited activity. Similar responses of the ATPase activity were observed with CydDC reconstituted into E. coli lipids. These results suggest that heme may have a regulatory role in CydDC-mediated transmembrane thiol transport

Formation of rectifier with gold nanoclusters

Gold nanoclusters encapsulated with organic molecules are of great interest for its possible applications in the fields of molecular electronics, catalysis and medical science. Here we demonstrate that monolayer and bilayer films of thiol-capped gold nanoclusters can exhibit diode-like properties provided controlled spatial asymmetry exist between two tunnel junctions used to connect a thiol capped gold nanoclusters. Current-voltage characteristics of this rectifier were obtained from conducting probe atomic force microscopy measurements and also from conventional two probe resistance measurements. Systematic x-ray reflectivity and atomic force microscopy measurements were carried out to characterize the spatial asymmetry introduced by a monolayer of fatty acid salt gadolinium stearate used to deposit thiol-capped gold nanocluster molecules on hydrophilic SiO2-Si(001) substrate by Langmuir Blodgett technique. This information was used to explain prominent rectification observed in these nano-structured films.Comment: 13 pages, 3 figure

Transport Studies of Isolated Molecular Wires in Self-Assembled Monolayer Devices

We have fabricated a variety of novel molecular diodes based on self-assembled-monolayers (SAM) of solid-state mixture of molecular wires (1,4 benzene-dimethane-thiol), and molecular insulator spacers (1-pentanethiol) with different concentration ratios r of wires/spacers, which were sandwiched between two gold (Au) electrodes. We introduce two new methods borrowed from Surface Science to (i) confirm the connectivity between the benzene-dimethane-thiol molecules with the upper Au electrode, and (ii) count the number of isolated molecular wires in the devices. The electrical transport properties of the SAM diodes were studied at different temperatures via the conductance and differential conductance spectra. We found that a potential barrier caused by the spatial connectivity gap between the pentanethiol molecules and the upper Au electrode dominates the transport properties of the pure pentanethiol SAM diode (r = 0). The transport properties of molecular diodes with low r-values are dominated by the conductance of the isolated benzene-dimethane-thiol molecules in the device. We found that the temperature dependence of the molecular diodes is much weaker than that of the pure pentanethiol device indicating the importance of the benzene-dimethane-thiol simultaneous bonding to the two Au electrodes that facilitate electrical transport. From the differential conductance spectra we also found that the energy difference, Delta between the Au electrode Fermi-level and the benzene-dimethane-thiol HOMO (or LUMO) level is ~1.5 eV; whereas it is ~2.5 eV for the pentanethiol molecule. The weak temperature dependent transport that we obtained for the SSM diodes reflects the weak temperature dependence of Delta.Comment: 38 p 8 Fi

Inelastic Tunneling Spectroscopy of Gold-Thiol and Gold-Thiolate Interfaces in Molecular Junctions: The Role of Hydrogen

It is widely believed that when a molecule with thiol (S-H) end groups bridges a pair of gold electrodes, the S atoms bond to the gold and the thiol H atoms detach from the molecule. However, little is known regarding the details of this process, its time scale, and whether molecules with and without thiol hydrogen atoms can coexist in molecular junctions. Here we explore theoretically how inelastic tunneling spectroscopy (IETS) can shed light on these issues. We present calculations of the geometries, low bias conductances and IETS of propanedithiol and propanedithiolate molecular junctions with gold electrodes. We show that IETS can distinguish between junctions with molecules having no, one or two thiol hydrogen atoms. We find that in most cases the single-molecule junctions in the IETS experiment of Hihath et al. [Nano Lett. 8, 1673 (2008)] had no thiol H atoms, but that a molecule with a single thiol H atom may have bridged their junction occasionally. We also consider the evolution of the IETS spectrum as a gold STM tip approaches the intact S-H group at the end of a molecule bound at its other end to a second electrode. We predict the frequency of a vibrational mode of the thiol H atom to increase by a factor \sim 2 as the gap between the tip and molecule narrows. Therefore, IETS should be able to track the approach of the tip towards the thiol group of the molecule and detect the detachment of the thiol H atom from the molecule when it occurs.Comment: 11 pages, 7 figures, 1 tabl

Myricetin, the main flavonoid in Syzygium cumini leaf, is a novel inhibitor of platelet thiol isomerases PDI and ERp5

Background: Flavonoids have been characterized as a prominent class of compounds to treat thrombotic diseases through the inhibition of thiol isomerases. Syzygium cumini is a flavonoid-rich medicinal plant that contains myricetin and gallic acid. Little is known about the potential anti-platelet properties of S. cumini and its constituent flavonoids. Objective To evaluate the anti-platelet effects and mechanism of action of a polyphenol-rich extract (PESc) from S. cumini leaf and its most prevalent polyphenols, myricetin and gallic acid. Methods PESc, myricetin and gallic acid were incubated with platelet-rich plasma and washed platelets to assess platelet aggregation and activation. In vitro platelet adhesion and thrombus formation as well as in vivo bleeding time were performed. Finally, myricetin was incubated with recombinant thiol isomerases to assess its potential to bind and inhibit these, whilst molecular docking studies predicted possible binding sites. Results: PESc decreased platelet activation and aggregation induced by different agonists. Myricetin exerted potent anti-platelet effects, whereas gallic acid did not. Myricetin reduced the ability of platelets to spread on collagen, form thrombi in vitro without affecting haemostasis in vivo. Fluorescence quenching studies suggested myricetin binds to different thiol isomerases with similar affinity, despite inhibiting only protein disulphide isomerase (PDI) and ERp5 reductase activities (IC50~3.5 μM). Finally, molecular docking studies suggested myricetin formed non-covalent bonds with PDI and ERp5. Conclusions: PESc and its most abundant flavonoid myricetin strongly inhibit platelet function. Additionally, myricetin is a novel inhibitor of ERp5 and PDI, unveiling a new therapeutic perspective for the treatment of thrombotic disorders