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The role of odorant- and pheromone-binding proteins (OBPs) in olfactory function is not fully understood. We found an OBP sequence from the stable fly, Stomoxys calcitrans , ScalOBP60, that has a 25 amino acid N-terminal extension with a high content of histidine and acidic amino acids, suggesting a possible metal binding activity. A search of public databases revealed a large number of other fly OBPs with histidine-rich N-terminal extensions, as well as beetle, wasp and ant OBPs with histidine-rich C-terminal extensions. We recombinantly expressed ScalOBP60, as well as a truncated sequence which lacks the histidine-rich N-terminal region, tScalOBP60. Using fluorescence quenching and electrospray quadrupole time-of-flight mass spectrometry (ESI-QTOF), we detected two different types of metal-binding sites. Divalent copper, nickel and zinc bind to the N-terminal histidine-rich region, and divalent copper binds to an internal sequence position. Comparison of the ESI-QTOF spectra of ScalOBP60 and tScalOBP60 showed that the histidine-rich sequence is structurally disordered, but it becomes more ordered in the presence of divalent metal. When copper is bound to the internal site, binding of a hydrophobic ligand to ScalOBP60 is inhibited. The internal and N-terminal metal sites interact allosterically, possibly through a conformational equilibrium, suggesting a mechanism for metal regulation of ligand binding to ScalOBP60. Based on our studies of ScalOBP60, we propose several possible olfactory and non-olfactory functions for this OBP. [Display omitted] • Many insect odorant-binding proteins have histidine-rich N- or C-terminal sequences. • Stable fly His-rich odorant-binding protein ScalOBP60 binds Cu2+, Zn2+, and Ni2+. • Truncated ScalOBP60, lacking histidine-rich sequence, binds Cu2+. • Cu2+ bound to truncated ScalOBP60 inhibits hydrophobic ligand binding. • Model proposed for metal ion regulation of hydrophobic ligand binding to ScalOBP60. [ABSTRACT FROM AUTHOR]

Cysteamine dioxygenase (ADO) has been reported to exhibit two distinct biological functions with a nonheme iron center. It catalyzes oxidation of both cysteamine in sulfur metabolism and N-terminal cysteine-containing proteins or peptides, such as regulator of G protein signaling 5 (RGS5). It thereby preserves oxygen homeostasis in a variety of physiological processes. However, little is known about its catalytic center and how it interacts with these two types of primary substrates in addition to O2. Here, using electron paramagnetic resonance (EPR), Mössbauer, and UV-visible spectroscopies, we explored the binding mode of cysteamine and RGS5 to human and mouse ADO proteins in their physiologically relevant ferrous form. This characterization revealed that in the presence of nitric oxide as a spin probe and oxygen surrogate, both the small molecule and the peptide substrates coordinate the iron center with their free thiols in a monodentate binding mode, in sharp contrast to binding behaviors observed in other thiol dioxygenases. We observed a substrate-bound B-type dinitrosyl iron center complex in ADO, suggesting the possibility of dioxygen binding to the iron ion in a side-on mode. Moreover, we observed substrate-mediated reduction of the iron center from ferric to the ferrous oxidation state. Subsequent MS analysis indicated corresponding disulfide formation of the substrates, suggesting that the presence of the substrate could reactivate ADO to defend against oxidative stress. The findings of this work contribute to the understanding of the substrate interaction in ADO and fill a gap in our knowledge of the substrate specificity of thiol dioxygenases. [ABSTRACT FROM AUTHOR]

Noble-metal nanoparticles have had a substantial impact across a diverse range of fields, including catalysis, sensing, photochemistry, optoelectronics, energy conversion and medicine. Although silver has very desirable physical properties, good relative abundance and low cost, gold nanoparticles have been widely favoured owing to their proved stability and ease of use. Unlike gold, silver is notorious for its susceptibility to oxidation (tarnishing), which has limited the development of important silver-based nanomaterials. Despite two decades of synthetic efforts, silver nanoparticles that are inert or have long-term stability remain unrealized. Here we report a simple synthetic protocol for producing ultrastable silver nanoparticles, yielding a single-sized molecular product in very large quantities with quantitative yield and without the need for size sorting. The stability, purity and yield are substantially better than those for other metal nanoparticles, including gold, owing to an effective stabilization mechanism. The particular size and stoichiometry of the product were found to be insensitive to variations in synthesis parameters. The chemical stability and structural, electronic and optical properties can be understood using first-principles electronic structure theory based on an experimental single-crystal X-ray structure. Although several structures have been determined for protected gold nanoclusters, none has been reported so far for silver nanoparticles. The total structure of a thiolate-protected silver nanocluster reported here uncovers the unique structure of the silver thiolate protecting layer, consisting of Ag2S5 capping structures. The outstanding stability of the nanoparticle is attributed to a closed-shell 18-electron configuration with a large energy gap between the highest occupied molecular orbital and the lowest unoccupied molecular orbital, an ultrastable 32-silver-atom excavated-dodecahedral core consisting of a hollow 12-silver-atom icosahedron encapsulated by a 20-silver-atom dodecahedron, and the choice of protective coordinating ligands. The straightforward synthesis of large quantities of pure molecular product promises to make this class of materials widely available for further research and technology development. [ABSTRACT FROM AUTHOR]

Mass spectrometry has played a key role in identifying the members of a series of gold clusters, which has enabled the development of magic-number duster theory. The successes of the gold duster system have yet to be repeated in another metal duster system, however. Silver dusters in particular have proven to be challenging due to their relative instability compared with gold clusters. Using the well- characterized gold nanocluster, Au25(SG)18, we present optimized electhspray ionizabon mass spectrometry (ESI-MS) instrumental parameters for the maximal transmission of the intact cluster. Parameters shown to have the largest effect on intact cluster transmission/detection include trap and transfer collision energy, source temperature, and cone gas flow rate. Herein we describe a general strategy to acquire mass spectra of fragile metal clusters with reliable mass assignments. By also optimizing sample solution conditions, high-quality ESI mass spectra of a prototypical silver:glutathione (Ag:SG) duster were obtained without significant fragmentation. By using gentle conditions and solution conditions designed to stabilize the clusters, fragmentation was dramatically reduced and mass spectra with isotopic resolution were measured. Using this strategy, we have made the first formula assignment for a ligand-protected Ag duster of Ag32(SG)19. [ABSTRACT FROM AUTHOR]

Abstract: We present the first complete amino acid sequences of Snow leopard (Uncia uncia) and Amur tiger (Panthera tigris altaica) hemoglobin (Hb) determined using a combined ESI MS and X-ray crystallography approach. Using mass spectrometry alone, we were able to achieve 81%, 90%, 87% and 72% sequence coverage for the α-, and β-globins of Snow leopard Hb and the α-, and β-chain of Amur tiger Hb, respectively. The incomplete mass spectrometry-determined sequences were used to process X-ray diffraction data of Snow leopard and Amur tiger Hb crystals solved to 2.1Å and 1.8Å, respectively. Using tandem mass spectrometry in concert with the electron density maps from X-ray structures, we were able to not only achieve 100% coverage of all amino acid sequences, but to also distinguish between isomeric (leucine and isoleucine) and isobaric (glutamine and lysine) residues. These results demonstrate the synergy between mass spectrometry and X-ray crystallography for the determination of full amino acid sequence coverage of abundant proteins. [Copyright &y& Elsevier]

23-Hydroxy ursolic acid (23-OH UA) is a potent atheroprotective and anti-obesogenic phytochemical, with anti-inflammatory and inflammation-resolving properties. In this study, we examined whether dietary 23-OH UA protects mice against the acute onset and progression of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). Female C57BL/6 mice were fed either a defined low-calorie maintenance diet (MD) or an MD supplemented with 0.2% wgt/wgt 23-OH UA for 5 weeks prior to actively inducing EAE and during the 30 days post-immunization. We observed no difference in the onset of EAE between the groups of mice, but ataxia and EAE disease severity were suppressed by 52% and 48%, respectively, and disease incidence was reduced by over 49% in mice that received 23-OH UA in their diet. Furthermore, disease-associated weight loss was strikingly ameliorated in 23-OH UA-fed mice. ELISPOT analysis showed no significant differences in frequencies of T cells producing IL-17 or IFN-γ between 23-OH UA-fed mice and control mice, suggesting that 23-OH UA does not appear to regulate peripheral T cell responses. In summary, our findings in EAE mice strongly suggest that dietary 23-OH UA may represent an effective oral adjunct therapy for the prevention and treatment of relapsing-remitting MS.

Profiling the repertoire of proteins associated with a given mRNA during the cell cycle is unstudied. Furthermore, it is easier to ask and answer what mRNAs a specific protein might bind to than the other way around. Here, we implemented an RNA-centric proximity labeling technology at different points in the cell cycle in highly synchronous yeast cultures. To understand how the abundance of FAS1 , encoding fatty acid synthase, peaks late in the cell cycle, we identified proteins that interact with the FAS1 transcript in a cell cycle-dependent manner. We used dCas13d-APEX2 fusions to target FAS1 and label nearby proteins, which were then identified by mass spectrometry. The glycolytic enzyme Tdh3p, a known RNA-binding protein, interacted with the FAS1 mRNA, and it was necessary for the periodic abundance of Fas1p in the cell cycle. These results point to unexpected connections between major metabolic pathways. They also underscore the role of mRNA-protein interactions for gene expression during cell division.

Cysteine dioxygenase (CDO) plays an essential role in sulfur metabolism by regulating homeostatic levels of cysteine. Human CDO contains a post-translationally generated Cys93-Tyr157 cross-linked cofactor. Here, we investigated this Cys-Tyr cross-linking by incorporating unnatural tyrosines in place of Tyr157 via a genetic method. The catalytically active variants were obtained with a thioether bond between Cys93 and the halogen-substituted Tyr157, and we determined the crystal structures of both wild-type and engineered CDO variants in the purely uncross-linked form and with a mature cofactor. Along with mass spectrometry and 19 F NMR, these data indicated that the enzyme could catalyze oxidative C-F or C-Cl bond cleavage, resulting in a substantial conformational change of both Cys93 and Tyr157 during cofactor assembly. These findings provide insights into the mechanism of Cys-Tyr cofactor biogenesis and may aid the development of bioinspired aromatic carbon-halogen bond activation.

Cysteamine dioxygenase (ADO) is a thiol dioxygenase whose study has been stagnated by the ambiguity as to whether or not it possesses an anticipated protein-derived cofactor. Reported herein is the discovery and elucidation of a Cys-Tyr cofactor in human ADO, crosslinked between Cys220 and Tyr222 through a thioether (C-S) bond. By genetically incorporating an unnatural amino acid, 3,5-difluoro-tyrosine (F 2 -Tyr), specifically into Tyr222 of human ADO, an autocatalytic oxidative carbon-fluorine bond activation and fluoride release were identified by mass spectrometry and 19 F NMR spectroscopy. These results suggest that the cofactor biogenesis is executed by a powerful oxidant during an autocatalytic process. Unlike that of cysteine dioxygenase, the crosslinking results in a minimal structural change of the protein and it is not detectable by routine low-resolution techniques. Finally, a new sequence motif, C-X-Y-Y(F), is proposed for identifying the Cys-Tyr crosslink., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

7α-Hydroxypregnenolone is an endogenous neuroactive steroid that stimulates locomotor activity. A synthesis of 7α-hydroxypregnenolone from pregnenolone, which takes advantage of an orthogonal protecting group strategy, is described. In detail, the C7-position was oxidized with CrO 3 and 3,5-dimethylpyrazole to yield a 7-keto steroid intermediate. The resulting 7-ketone was stereoselectively reduced to the 7α-hydroxy group with lithium tri-sec-butylborohydride. In contrast, reduction of the same 7-ketone intermediate with NaBH 4 resulted in primarily the 7β-hydroxy epimer. Furthermore, in an alternative route to the target compound, the 7α-hydroxy group was successfully incorporated by direct C-H allylic benzoyloxylation of pregnenolone-3-acetate with CuBr and tert-butyl peroxybenzoate followed by saponification. The disclosed syntheses to 7-oxygenated steroids are amenable to potentially obtain other biologically active sterols and steroids., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The shape-dependent activity of gold nanoparticles (AuNP) was studied by testing them as electrocatalysts for the notoriously slow non-enzymatic oxidation of glucose in neutral solutions. The AuNP of spherical and irregular (including polyhedral) morphologies were synthesized and attached to glassy carbon electrodes with chitosan. Voltammetric and mass spectrometric studies showed that the irregular AuNP were more catalytically active toward the oxidation of glucose to gluconic acid. No obvious differences between both morphologies were found based on their X-ray diffraction patterns and HRTEM images suggesting that the crystallographic orientation alone did not account for their catalytic properties. While both morphologies contain the (111) crystallographic planes that are catalytic toward glucose oxidation, the better activity of irregular AuNP was ascribed to a higher surface density of incipient gold oxide acting as a fast redox mediator for glucose oxidation. Supporting this, the AuNP of both morphologies oxidized glucose after their anodic activation, although not to the same extent. The amperometric (0.30 V) determination of glucose at electrodes made of irregular AuNP yielded a wide linear calibration plot (0.20-110 mM; R 2 , 0.998), sensitivity of 66 μA M -1 cm -2 , limit of detection of 100 μM (S/N, 3), and a response time below 5 s. The advantage of low-cost irregular AuNP over macro gold is that they are catalytic toward glucose oxidation without any need for prior activation.

The in vitro deuteroacetylation of histones obtained from biological sources has been used previously in bottom-up mass spectrometry analyses to quantitate the percent of endogenous acetylation of specific lysine sites and/or peptides. In this report, derivatization of unmodified lysine residues on histones is used in combination with high performance mass spectrometry, including combined HPLC MS/MS, to distinguish and quantitate endogenously acetylated isoforms occurring within the same tryptic peptide sequence and to extend this derivatization strategy to other post-translational modifications, specifically methylation, dimethylation and trimethylation. The in vitro deuteroacetylation of monomethylated lysine residues is observed, though dimethylated or trimethylated residues are not derivatised. Comparison of the relative intensities ascribed to the deuteroacetylated and monomethylated species with the deuteroacetylated but unmethylated analog, provides an opportunity to estimate the percent of methylation at that site. In addition to the observed fragmentation patterns, the very high mass accuracy available on the Orbitrap mass spectrometer can be used to confirm the structural isoforms, and in particular to distinguish between trimethylated and acetylated species.