Dimethyl disulfide dissociation is both ultrafast and very slow.
Abstract not provided.
Cole-Filipiak, Neil↗
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Chamber studies of OH + dimethyl sulfoxide and dimethyl disulfide: insights into the dimethyl sulfide oxidation mechanism
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54 ENVIRONMENTAL SCIENCES↗
Thiol-based pathways in the thylakoid lumen and their role in photoprotection
The long-term goal of this research is to elaborate the catalysis of thiol-disulfide transactions in the thylakoid lumen, a compartment required for transducing energy via photophosphorylation. The molecular identity of the redox players catalyzing thiol-disulfide exchanges, their relevant targets of action in vivo and how thiol-disulfide chemistry in general controls photosynthesis are the experimental questions this research aims to address. Through previous work, we documented the requirement of catalyzed disulfide formation and reduction for the biogenesis of two photosynthetic enzymes, namely Photosystem II and the cytochrome b6f complex. We established that dedicated trans-thylakoid pathways operate in these processes by delivering reducing and oxidizing power to cysteine-containing subunits of the photosynthetic complexes, which are localized in the lumen compartment. Here we focused on CCDA, CCS4 and CCS5/HCF164, the components of the disulfide-reducing pathway which maintains the heme binding cysteines of apoforms of plastid cytochrome c in the reduced form prior to covalent heme ligation. We showed that CCS4 and CCS5 operate in functionally redundant pathways for the supply of reducing power and postulate a role for CCS4 in recruiting the source of reductants to CCDA or controlling the CCDA-dependent transduction of reducing equivalents. We also demonstrate the disulfide reducing pathway operates to counter thiol oxidation of the heme-binding cysteines by disulfide forming enzyme LTO1 in the thylakoid lumen.
59 BASIC BIOLOGICAL SCIENCES↗
Three-dimensional hierarchically porous MoS 2 foam as high-rate and stable lithium-ion battery anode
Architected materials that actively respond to external stimuli hold tantalizing prospects for applications in energy storage, wearable electronics, and bioengineering. Molybdenum disulfide, an excellent two-dimensional building block, is a promising candidate for lithium-ion battery anode. However, the stacked and brittle two-dimensional layered structure limits its rate capability and electrochemical stability. Here we report the dewetting-induced manufacturing of two-dimensional molybdenum disulfide nanosheets into a three-dimensional foam with a structural hierarchy across seven orders of magnitude. Our molybdenum disulfide foam provides an interpenetrating network for efficient charge transport, rapid ion diffusion, and mechanically resilient and chemically stable support for electrochemical reactions. These features induce a pseudocapacitive energy storage mechanism involving molybdenum redox reactions, confirmed by in-situ X-ray absorption near edge structure. The extraordinary electrochemical performance of molybdenum disulfide foam outperforms most reported molybdenum disulfide-based Lithium-ion battery anodes and state-of-the-art materials. This work opens promising inroads for various applications where special properties arise from hierarchical architecture.
25 ENERGY STORAGE↗
Conotoxin Prediction: New Features to Increase Prediction Accuracy
Conotoxins are toxic, disulfide-bond-rich peptides from cone snail venom that target a wide range of receptors and ion channels with multiple pathophysiological effects. Conotoxins have extraordinary potential for medical therapeutics that include cancer, microbial infections, epilepsy, autoimmune diseases, neurological conditions, and cardiovascular disorders. Despite the potential for these compounds in novel therapeutic treatment development, the process of identifying and characterizing the toxicities of conotoxins is difficult, costly, and time-consuming. This challenge requires a series of diverse, complex, and labor-intensive biological, toxicological, and analytical techniques for effective characterization. While recent attempts, using machine learning based solely on primary amino acid sequences to predict biological toxins (e.g., conotoxins and animal venoms), have improved toxin identification, these methods are limited due to peptide conformational flexibility and the high frequency of cysteines present in toxin sequences. This results in an enumerable set of disulfide-bridged foldamers with different conformations of the same primary amino acid sequence that affect function and toxicity levels. Consequently, a given peptide may be toxic when its cysteine residues form a particular disulfide-bond pattern, while alternative bonding patterns (isoforms) or its reduced form (free cysteines with no disulfide bridges) may have little or no toxicological effects. Similarly, the same disulfide-bond pattern may be possible for other peptide sequences and result in different conformations that all exhibit varying toxicities to the same receptor or to different receptors. We present here new features, when combined with primary sequence features to train machine learning algorithms to predict conotoxins, that significantly increase prediction accuracy.
collisional cross section↗
The SrrAB two-component system regulates Staphylococcus aureus pathogenicity through redox sensitive cysteines
Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. In this work, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.
59 BASIC BIOLOGICAL SCIENCES↗
Torsional twist of the SARS ‐ CoV and SARS ‐ CoV ‐2 SUD ‐N and SUD ‐M domains
Abstract Coronavirus non‐structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vesicle that houses the replication–transcription complex. Nsp3 in SARS‐like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD‐N (Macrodomain 2) and SUD‐M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x‐ray structure of SARS‐CoV SUD‐N/M as well as a mutant variant of SARS‐CoV‐2 SUD‐N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD‐N and SUD‐M are not rigidly associated but rather have significant rotational flexibility. Phylogenetic analysis supports that the potential to form the disulfide bond is common across betacoronavirus isolates from many bat species and civets, but also one or both of the cysteines that form the disulfide bond are absent across isolates from bats and pangolins. The absence of these cysteines does not impact viral replication or protein translation.
Rosas‐Lemus, Monica [Department of Microbiology‐Im↗
Modifying the resolving cysteine affects the structure and hydrogen peroxide reactivity of peroxiredoxin 2
Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H 2 O 2 and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulfide formation, affect H 2 O 2 reactivity. To investigate, we designed mutants to cause minimal (C172S) or substantial (C172D and C172W) structural disruption. Stopped flow kinetics and mass spectrometry showed that mutation to Ser had minimal effect on rates of oxidation and hyperoxidation, whereas Asp and Trp decreased both by 100-fold. To relate to structural changes, we solved the crystal structures of reduced WT and C172S Prdx2. The WT structure is highly similar to that of the published hyperoxidized form. C172S is closely related but more flexible and as demonstrated by size exclusion chromatography and analytical ultracentrifugation, a weaker decamer. Size exclusion chromatography and analytical ultracentrifugation showed that the C172D and C172W mutants are also weaker decamers than WT, and small-angle X-ray scattering analysis indicated greater flexibility with partially unstructured regions consistent with C-terminal unfolding. We propose that these structural changes around C172 negatively impact the active site geometry to decrease reactivity with H 2 O 2 . This is relevant for Prdx turnover as intermediate mixed disulfides with C172 would also be disruptive and could potentially react with peroxides before resolution is complete.
59 BASIC BIOLOGICAL SCIENCES↗
Structural and mechanistic basis for redox sensing by the cyanobacterial transcription regulator RexT
Abstract Organisms have a myriad of strategies for sensing, responding to, and combating reactive oxygen species, which are unavoidable consequences of aerobic life. In the heterocystous cyanobacterium Nostoc sp. PCC 7120, one such strategy is the use of an ArsR-SmtB transcriptional regulator RexT that senses H 2 O 2 and upregulates expression of thioredoxin to maintain cellular redox homeostasis. Different from many other members of the ArsR-SmtB family which bind metal ions, RexT has been proposed to use disulfide bond formation as a trigger to bind and release DNA. Here, we present high-resolution crystal structures of RexT in the reduced and H 2 O 2 -treated states. These structures reveal that RexT showcases the ArsR-SmtB winged-helix-turn-helix fold and forms a vicinal disulfide bond to orchestrate a response to H 2 O 2 . The importance of the disulfide-forming Cys residues was corroborated using site-directed mutagenesis, mass spectrometry, and H 2 O 2 -consumption assays. Furthermore, an entrance channel for H 2 O 2 was identified and key residues implicated in H 2 O 2 activation were pinpointed. Finally, bioinformatics analysis of the ArsR-SmtB family indicates that the vicinal disulfide “redox switch” is a unique feature of cyanobacteria in the Nostocales order, presenting an interesting case where an ArsR-SmtB protein scaffold has been evolved to showcase peroxidatic activity and facilitate redox-based regulation.
54 ENVIRONMENTAL SCIENCES↗
Accurate Free Energies for Complex Condensed-Phase Reactions Using an Artificial Neural Network Corrected DFTB/MM Methodology
Semiempirical methods like density functional tight-binding (DFTB) allow extensive phase space sampling, making it possible to generate free energy surfaces of complex reactions in condensed-phase environments. Such a high efficiency often comes at the cost of reduced accuracy, which may be improved by developing a specific reaction parametrization (SRP) for the particular molecular system. Thiol–disulfide exchange is a nucleophilic substitution reaction that occurs in a large class of proteins. Its proper description requires a high-level ab initio method, while DFT-GAA and hybrid functionals were shown to be inadequate, and so is DFTB due to its DFT-GGA descent. We develop an SRP for thiol–disulfide exchange based on an artificial neural network (ANN) implementation in the DFTB+ software and compare its performance to that of a standard SRP approach applied to DFTB. Furthermore, as an application, we use both new DFTB-SRP as components of a QM/MM scheme to investigate thiol–disulfide exchange in two molecular complexes: a solvated model system and a blood protein. Demonstrating the strengths of the methodology, highly accurate free energy surfaces are generated at a low cost, as the augmentation of DFTB with an ANN only adds a small computational overhead.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Chlorophyllase from Arabidopsis thaliana Reveals an Emerging Model for Controlling Chlorophyll Hydrolysis
Chlorophyll (Chl) is one of Nature’s most complex pigments to biosynthesize and derivatize. This pigment is vital for survival and also paradoxically toxic if overproduced or released from a protective protein scaffold. Therefore, along with the mass production of Chl, organisms also invest in mechanisms to control its degradation and recycling. One important enzyme that is involved in these latter processes is chlorophyllase. This enzyme is employed by numerous photosynthetic organisms to hydrolyze the phytol tail of Chl. Although traditionally thought to catalyze the first step of Chl degradation, recent work suggests that chlorophyllase is instead employed during times of abiotic stress or conditions that produce reactive oxygen species. However, the molecular details regarding how chlorophyllases are regulated to function under such conditions remain enigmatic. Here, we investigate the Arabidopsis thaliana chlorophyllase isoform AtCLH2 using site-directed mutagenesis, mass spectrometry, dynamic light scattering, size-exclusion multiangle light scattering, and both steady-state enzyme kinetic and thermal stability measurements. Through these experiments, we show that AtCLH2 exists as a monomer in solution and contains two disulfide bonds. One disulfide bond putatively maps to the active site, whereas the other links two N-terminal Cys residues together. These disulfide bonds are cleaved by chemical or chemical and protein-based reductants, respectively, and are integral to maintaining the activity, stability, and substrate scope of the enzyme. This work suggests that Cys residue oxidation in chlorophyllases is an emerging regulatory strategy for controlling the hydrolysis of Chl pigments.
59 BASIC BIOLOGICAL SCIENCES↗
A cryptic oxidoreductase safeguards oxidative protein folding in Corynebacterium diphtheriae
In many gram-positive Actinobacteria, including Actinomyces oris and Corynebacterium matruchotii , the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is essential for bacterial viability by an unidentified mechanism. Intriguingly, in Corynebacterium diphtheriae , the deletion of mdbA blocks cell growth only at 37 °C but not at 30 °C, suggesting the presence of alternative oxidoreductase enzyme(s). By isolating spontaneous thermotolerant revertants of the mdbA mutant at 37 °C, we obtained genetic suppressors, all mapped to a single T-to-G mutation within the promoter region of tsdA , causing its elevated expression. Strikingly, increased expression of tsdA —via suppressor mutations or a constitutive promoter—rescues the pilus assembly and toxin production defects of this mutant, hence compensating for the loss of mdbA . Structural, genetic, and biochemical analyses demonstrated TsdA is a membrane-tethered thiol-disulfide oxidoreductase with a conserved CxxC motif that can substitute for MdbA in mediating oxidative folding of pilin and toxin substrates. Together with our observation that tsdA expression is upregulated at nonpermissive temperature (40 °C) in wild-type cells, we posit that TsdA has evolved as a compensatory thiol-disulfide oxidoreductase that safeguards oxidative protein folding in C. diphtheriae against thermal stress.
59 BASIC BIOLOGICAL SCIENCES↗
OpenAWSEM with Open3SPN2: A fast, flexible, and accessible framework for large-scale coarse-grained biomolecular simulations
We present OpenAWSEM and Open3SPN2, new cross-compatible implementations of coarse-grained models for protein (AWSEM) and DNA (3SPN2) molecular dynamics simulations within the OpenMM framework. These new implementations retain the chemical accuracy and intrinsic efficiency of the original models while adding GPU acceleration and the ease of forcefield modification provided by OpenMM’s Custom Forces software framework. By utilizing GPUs, we achieve around a 30-fold speedup in protein and protein-DNA simulations over the existing LAMMPS-based implementations running on a single CPU core. We showcase the benefits of OpenMM’s Custom Forces framework by devising and implementing two new potentials that allow us to address important aspects of protein folding and structure prediction and by testing the ability of the combined OpenAWSEM and Open3SPN2 to model protein-DNA binding. The first potential is used to describe the changes in effective interactions that occur as a protein becomes partially buried in a membrane. We also introduced an interaction to describe proteins with multiple disulfide bonds. Using simple pairwise disulfide bonding terms results in unphysical clustering of cysteine residues, posing a problem when simulating the folding of proteins with many cysteines. We now can computationally reproduce Anfinsen’s early Nobel prize winning experiments by using OpenMM’s Custom Forces framework to introduce a multi-body disulfide bonding term that prevents unphysical clustering. Our protein-DNA simulations show that the binding landscape is funneled towards structures that are quite similar to those found using experiments. In summary, this paper provides a simulation tool for the molecular biophysics community that is both easy to use and sufficiently efficient to simulate large proteins and large protein-DNA systems that are central to many cellular processes. These codes should facilitate the interplay between molecular simulations and cellular studies, which have been hampered by the large mismatch between the time and length scales accessible to molecular simulations and those relevant to cell biology.
59 BASIC BIOLOGICAL SCIENCES↗
Cysteine residues contribute to the regulation of Arabidopsis state transition 7 kinase
State transitions are an acclimatory response by which plants, algae, and cyanobacteria counteract photosynthetic inefficiency caused by changes in incident light quality. In plants and green algae, state transition 7 (STN7/STT7) kinase promotes state 2 transition. Conserved cysteine residues are implicated in STN7/STT7 regulation, but the precise nature of their involvement remains unclear. Here, an analysis of the STN7 thiols in vitro and a determination of their midpoint redox potential indicate that the lumenal disulfide linkage is unlikely to be redox regulated while the stromal cysteines form a regulatory intramolecular disulfide. We further show that thioredoxin f 1 (Trx‐ f 1) reduces the STN7 stromal disulfide linkage as consistent with a Trx‐ f 1‐mediated inhibition of the kinase under high light.
Ibrahim, Iskander M.↗
Light-Mediated Synthesis and Reprocessing of Dynamic Bottlebrush Elastomers under Ambient Conditions
We introduce a novel grafting-through polymerization strategy to synthesize dynamic bottlebrush polymers and elastomers in one step using light to construct a disulfide-containing backbone. The key starting material—α-lipoic acid (LA)—is commercially available, inexpensive, and biocompatible. When installed on the chain end(s) of poly(dimethylsiloxane) (PDMS), the cyclic disulfide unit derived from LA polymerizes under ultraviolet (UV) light in ambient conditions. Significantly, no additives such as initiator, solvent, or catalyst are required for efficient gelation. Formulations that include bis-LA-functionalized cross-linker yield bottlebrush elastomers with high gel fractions (83–98%) and tunable, supersoft shear moduli in the ~20–200 kPa range. An added advantage of these materials is the dynamic disulfide bonds along each bottlebrush backbone, which allow for light-mediated self-healing and on-demand chemical degradation. These results highlight the potential of simple and scalable synthetic routes to generate unique bottlebrush polymers and elastomers based on PDMS.
36 MATERIALS SCIENCE↗
Identification and structural analysis of a thermophilic β-1,3-glucanase from compost
β-1,3-glucanase can specifically hydrolyze glucans to oligosaccharides and has potential applications in biotechnology. We used the metatranscriptomic technology to discover a thermophilic β-1,3-glucanase from compost. The phylogenetic study shows that it belongs to the family 16 glycoside hydrolase (GH16) and is most homologous with an enzyme from Streptomyces sioyaensis, an actinobacterium. It has the activity of 146.9 U/mg in the optimal reaction condition (75 °C and pH 5.5). Its catalytic domain was crystallized and diffracted to 1.14 Å resolution. The crystal structure shows a sandwich-like β-jelly-roll fold with two disulfide bonds. After analyzing the occurring frequencies of these cysteine residues, we designed two mutants (C160G and C180I) to study the role of these disulfide bonds. Both mutants have decreased their optimal temperature from 75 to 70 °C, which indicate that the disulfide bonds are important to maintain thermostability. Interestingly, the activity of C160G has increased ~ 17% to reach 171.4 U/mg. We speculate that the increased activity of C160G mutant is due to increased dynamics near the active site. Our studies give a good example of balancing the rigidity and flexibility for enzyme activity, which is helpful for protein engineering.
59 BASIC BIOLOGICAL SCIENCES↗
An ultralong CDRH2 in HCV neutralizing antibody demonstrates structural plasticity of antibodies against E2 glycoprotein
A vaccine protective against diverse HCV variants is needed to control the HCV epidemic. Structures of E2 complexes with front layer-specific broadly neutralizing antibodies (bNAbs) isolated from HCV-infected individuals, revealed a disulfide bond-containing CDRH3 that adopts straight (individuals who clear infection) or bent (individuals with chronic infection) conformation. To investigate whether a straight versus bent disulfide bond-containing CDRH3 is specific to particular HCV-infected individuals, we solved a crystal structure of the HCV E2 ectodomain in complex with AR3X, a bNAb with an unusually long CDRH2 that was isolated from the chronically-infected individual from whom the bent CDRH3 bNAbs were derived. The structure revealed that AR3X utilizes both its ultralong CDRH2 and a disulfide motif-containing straight CDRH3 to recognize the E2 front layer. These results demonstrate that both the straight and bent CDRH3 classes of HCV bNAb can be elicited in a single individual, revealing a structural plasticity of VH1-69-derived bNAbs.
59 BASIC BIOLOGICAL SCIENCES↗
Evaluation of 186 WS 2 target material for production of high specific activity 186 Re via proton irradiation: separation, radiolabeling and recovery/recycling
Enriched tungsten disulfide ( 186 WS 2 ) was evaluated at increasing proton beam currents (20-50 µA) and times (up to 4 h) on a GE PETtrace cyclotron for production of high specific activity (HSA) 186 Re. The HSA 186 Re was separated from the irradiated target as [ 186 Re][ReO 4 ] - by a liquid-liquid extraction method and radiolabeled with a new N 2 S 2 ligand (222-MAMA-N-ethylpropionate). The enriched 186 W was recovered from the extraction process, analyzed for purity and enrichment, and converted back to the disulfide ( 186 WS 2 ). The results demonstrate that the 186 WS 2 is an easily pressed target material that can withstand relatively high currents and can be readily recovered and recycled. The 186 Re produced was isolated in high specific activity and readily formed the radiotracers [ 186 Re][ReO(222-MAMA-N ethylpropionate)] and [ 186 Re][Re(CO) 3 (OH 2 ) 3 ] + .
186Re,N2S2 ligand↗