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We present a sensitive, selective and robust method for the determination of 14 thiol compounds in aqueous samples. Thiols were derivatized with ω-bromoacetonylquinolinium bromide (BQB) and its deuterium labeled equivalent D7-ω-bromoacetonylquinolinium bromide (D7). Derivatized thiols were preconcentrated by online solid-phase extraction (SPE) followed by liquid chromatography separation and electrospray ionization tandem mass spectrometry determination (SPE/LC-ESI-MS/MS). The robustness of the method was validated for wide ranges in pH, salinity, and concentrations of sulfide and dissolved organic carbon (DOC) to cover contrasting natural water types. The limits of detection (LODs) for the thiols were 3.1-66 pM. Between 6 and 14 of the thiols were detected in different natural sample types at variable concentrations: boreal wetland porewater (0.7-51 nM), estuarine sediment porewater (50 pM-11 nM), coastal sea water (60 pM-16 nM), and sulfate reducing bacterium cultures (80 pM-4 nM). MS/MS fragmentation of the compounds produces two pairs of common product ions, m/z 130.2/137.1 and 218.1/225.1, which enables scanning for unknown thiols in precursor ion scan mode. Using this approach, we identified cysteine, mercaptoacetic acid, N-acetyl-L-cysteine and sulfurothioic S-acid in boreal wetland porewater. The performance of the developed method sets a new state of the art for the determination of thiol compounds in environmental and biological samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cells and metabolic products of Desulfovibrio desulfuricans were successfully used to separate quartz from hematite through environmentally benign microbially induced flotation. Bacterial metabolic products such as extracellular proteins and polysaccharides were isolated from both unadapted and mineral-adapted bacterial metabolite and their basic characteristics were studied in order to get insight into the changes brought about on bioreagents during adaptation. Interaction between bacterial cells and metabolites with minerals like hematite and quartz brought about significant surface-chemical changes on both the minerals. Quartz was rendered more hydrophobic, while hematite became more hydrophilic after biotreatment. The predominance of bacterial polysaccharides on interacted hematite and of proteins on quartz was responsible for the above surface-chemical changes, as attested through adsorption studies. Surface-chemical changes were also observed on bacterial cells after adaptation to the above minerals. Selective separation of quartz from hematite was achieved through interaction with quartz-adapted bacterial cells and metabolite. Mineral-specific proteins secreted by quartz-adapted cells were responsible for conferment of hydrophobicity on quartz resulting in enhanced separation from hematite through flotation., (2010 Elsevier B.V. All rights reserved.)

Flavodoxin adopts the common repeat beta/alpha topology and folds in a complex kinetic reaction with intermediates. To better understand this reaction, we analyzed a set of Desulfovibrio desulfuricans apoflavodoxin variants with point mutations in most secondary structure elements by in vitro and in silico methods. By equilibrium unfolding experiments, we first revealed how different secondary structure elements contribute to overall protein resistance to heat and urea. Next, using stopped-flow mixing coupled with far-UV circular dichroism, we probed how individual residues affect the amount of structure formed in the experimentally detected burst-phase intermediate. Together with in silico folding route analysis of the same point-mutated variants and computation of growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central beta-strand 3 (i.e., at beta-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in folding nuclei growth correlates with the in vitro burst-phase circular dichroism amplitude. In addition, phi-value analysis (in vitro and in silico) of the overall folding barrier to apoflavodoxin's native state revealed that native-like interactions in most of the beta-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central beta-sheet directs initial misfolding, while proper alignment on both sides of beta-strand 3 is necessary for productive folding., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The determination of methylmercury (MeHg) in environmental samples by ethylation derivation-gas chromatography-atomic fluorescence spectrometry (ED-GC-AFS) is associated with an intimate problem of water moisture accumulation introduced in the ethylation step, which enters the detection system and cause a spectroscopic interference. With a simple modification on the GC-AFS system, this problem was eliminated and the analytical quality of the measurements was significantly improved. The presence of dissolved sulfide in samples can also cause serious chemical interference in the ethylation step resulting in lower or total loss of the MeHg signal. It was found that a masking system of CuSO(4)-Na(2)C(2)O(4) was able to eliminate this interference. With this system, the accurate determination of trace amount of MeHg in high dissolved sulfide containing samples was achieved. Satisfactory analytical results were obtained with the certified reference sediment IAEA405, sulfate reducing bacteria culture and sulfide containing water samples. The limit of detection and quantitation of this masking system is 0.01 and 0.04ngL(-1) respectively. Other factors affecting ethylation are also discussed.

We have systematically explored the Hofmeister effects of cations and anions (0.3-1.75 M range) for acidic Desulfovibrio desulfuricans apoflavodoxin (net charge -19, pH 7) and basic horse heart cytochrome c (net charge +17, pH 4.5). The Hofmeister effect of the ions on protein thermal stability was assessed by the parameter dT trs/d[ion] (T trs; thermal midpoint). We show that dT trs/d[ion] correlates with ion partition coefficients between surface and bulk water and ion surface tension effects: this suggests direct interactions between ions and proteins. Surprisingly, the stability effects of the different ions on the two model proteins are similar, implying a major role of the peptide backbone, instead of charged groups, in mediation of the interactions. Upon assessing chemical/physical properties of the ions responsible for the Hofmeister effects on protein stability, ion charge density was identified as most important. Taken together, our study suggests key roles for ion hydration and the peptide group in facilitating interactions between Hofmeister ions and proteins.

Apoflavodoxin from the sulfate reducing bacteria Desulfovibrio desulfuricans is a small, acidic protein with a net charge of -19 at neutral pH. Here, we show that monovalent cations in biologically relevant amounts have dramatic effects on apoflavodoxin stability. The effect is largest for Gdm(+) and decreases as a function of increased cation charge density (Gdm(+)>NH(4)(+)K(+) approximately Cs(+) approximately Na(+)>Li(+)). A linear correlation of stabilizing effects with cation hydration properties suggests an important role of dehydration in efficient cation interaction with the protein. The effects on stability are due to preferential binding of one cation to native apoflavodoxin and results in an increase in thermal midpoint of 20 degrees C and the free energy of unfolding (at 20 degrees C) increases fivefold. Tuning of biophysical properties (such as folding and ligand/cofactor binding) of acidic proteins by cation binding may be important in vivo.

Folding of cofactor-binding proteins involves ligand binding in addition to polypeptide folding. We here assess the kinetic folding/binding landscape for Desulfovibrio desulfuricans flavodoxin that coordinates an FMN cofactor. The apo-form folds in a two-step process involving a burst-phase intermediate. Studies on Tyr98Ala and Trp60Ala variants reveal that these aromatics-that stack with the FMN in the holo-form-are not participating in the apo-protein folding pathway. However, these residues are essential for FMN interactions with the unfolded protein during refolding of holo-flavodoxin. Unfolding of wild-type holo-flavodoxin is coupled to FMN dissociation whereas for Tyr98Ala and Trp60Ala holo-variants, FMN dissociates before polypeptide unfolding. Both variants refold as apo-proteins before FMN rebinds. In sharp contrast, refolding of unfolded wild-type holo-flavodoxin is over an order of magnitude faster than that of the apo-form, the pathway does not include a burst-phase intermediate, and the speed is independent of FMN excess ratio. These observations demonstrate that FMN binds rapidly to the unfolded polypeptide and guides folding straight to the native state. As this path to functional D. desulfuricans holo-flavodoxin is faster than if the cofactor binds to pre-folded apo-protein, this is one of few examples where molecular recognition via a "fly-casting" mechanism is kinetically favored.

The structure of the type I tetraheme cytochrome c(3) from Desulfovibrio desulfuricans G20 was determined to 1.5 Angstrom by X-ray crystallography. In addition to the oxidized form, the structure of the molybdate-bound form of the protein was determined from oxidized crystals soaked in sodium molybdate. Only small structural shifts were obtained with metal binding, consistent with the remarkable structural stability of this protein. In vitro experiments with pure cytochrome showed that molybdate could oxidize the reduced cytochrome, although not as rapidly as U(VI) present as uranyl acetate. Alterations in the overall conformation and thermostability of the metal-oxidized protein were investigated by circular dichroism studies. Again, only small changes in protein structure were documented. The location of the molybdate ion near heme IV in the crystal structure suggested heme IV as the site of electron exit from the reduced cytochrome and implicated Lys14 and Lys56 in binding. Analysis of structurally conserved water molecules in type I cytochrome c(3) crystal structures identified interactions predicted to be important for protein stability and possibly for intramolecular electron transfer among heme molecules.