Search NASA⌕ Search

SEARCH · Search NASA

Results for “Cysteine”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Exploration of Nirmatrelvir Derivatives as Optimized SARS‐CoV‐2 Antivirals

Nirmatrelvir (NMV) is a SARS‐CoV‐2 antiviral component of the approved COVID‐19 therapeutic Paxlovid. It is a reversible covalent inhibitor of SARS‐CoV‐2 main protease (M Pro ) that is effluxed from human cells by P‐glycoprotein (P‐gp). To identify NMV analogs with improved potency and reduced P‐gp efflux, a structure–activity relationship campaign was conducted. Warheads alternative to nitrile for engaging the active site cysteine were tested showing aldehyde and dichloroacetamide with better enzyme inhibition potency. Crystal structure of MPI‐136−M Pro shows its aldehyde warhead forming a thiohemiacetal with active Cys145 of M Pro . Several S4 binders were explored revealing that an O‐to‐S shift at the N ‐terminal amide leads to better enzyme inhibition. By exploring different combinations of S2, S3, and S4 binders, two inhibitors with better enzyme inhibition potency than NMV were found. Crystal structure of MPI‐148, with ( S )‐2‐azaspiro[4,5]decane‐3‐carboxylate as an alternative S2 binder, shows extensive hydrogen‐bond networks for locking the inhibitor in active site, explaining high affinity of NMV analogs. Further characterization of cellular M Pro engagement and antiviral potency against SARS‐CoV‐2 revealed four inhibitors with greater potency than NMV in P‐gp‐expressing cells. Studies with the P‐gp inhibitor CP‐100356 showed that these compounds were less sensitive to P‐gp inhibition than NMV, consistent with reduced P‐gp‐mediated efflux.

Alugubelli, Yugendar R. [Texas A&M Drug Discovery ↗
DOE OSTI2020

Selective Enrichment Coupled with Proteomics to Identify S-Acylated Plasma Membrane Proteins in Arabidopsis

Protein S-acylation, predominately in the form of palmitoylation, is a reversible lipid post-translational modification (PTM) on cysteines that plays important roles in protein localization, trafficking, activity, and complex assembly. The functions and regulatory mechanisms of Sacylation have been extensively studied in mammals, owing to remarkable development of the high-resolution proteomics and the discovery of the S-acylation-related enzymes. However, the advance of S-acylation studies in plants lags farther behind than that in mammals, mainly due to the lack of knowledge about proteins responsible for this process, such as protein acyltransferases (PATs) and their substrates. In this article, a systematic protocol to study global S-acylation in Arabidopsis seedlings is described. The procedures are presented in detail, including preparation of Arabidopsis seedlings, enrichment of plasma membrane (PM) proteins, followed by enrichment of S-acylated proteins/peptides based on the acyl-biotin exchange (ABE) method, and large-scale identification of S-acylated proteins/peptides via mass spectrometry. This approach enables researchers to study S-acylation of PM proteins in plants in a systematic and straightforward way. Basic protocol 1: Preparation of Arabidopsis seedling materials Basic protocol 2: Isolation and enrichment of plasma membrane proteins Support protocol 1: Determination of protein concentration using BCA assay Basic Protocol 3: Enrichment of S-acylated proteins by ABE method Support protocol 2: Protein precipitation by methanol/chloroform method Basic Protocol 4: Trypsin digestion and proteomic analysis Alternate protocol 1: Pre-resin digestion and peptide level enrichment.

S-acylation, palmitoylation, plasma membrane prote↗
DOE OSTI2020

Solution structure for an Encephalitozoon cuniculi adrenodoxin-like protein in the oxidized state

Encephalitozoon cuniculi is a unicellular, obligate intracellular eukaryotic parasite in the Microsporidia family and one of the agents responsible for microsporidosis infections in humans. Like most Microsporidia, the genome of E. cuniculi is markedly reduced and the organism contains mitochondria-like organelles called mitosomes instead of mitochondria. Here we report the solution NMR structure for a protein physically associated with mitosome-like organelles in E. cuniculi, the 128-residue, adrenodoxin-like protein Ec-Adx (UniProt ID Q8SV19) in the [2Fe-2S] ferredoxin superfamily. Oxidized Ec-Adx contains a mixed four-strand β-sheet, β2-β1-β4-β3 (↓↑↑↓), loosely encircled by three α-helices and two 310-helices. This fold is similar to the structure observed in other adrenodoxin and adrenodoxin-like proteins except for the absence of a fifth anti-parallel β-strand next to β3 and the position of α3. Cross peaks are missing or cannot be unambiguously assigned for 20 amide resonances in the 1 H- 15 N HSQC spectrum of Ec-Adx. These missing residues are clustered primarily in two regions, G48-V61 and L94-L98, containing the four cysteine residues predicted to ligate the paramagnetic [2Fe-2S] cluster. Missing amide resonances in 1 H- 15 N HSQC spectra are detrimental to NMR-based solution structure calculations because 1 H- 1 H NOE restraints are absent (glass half-empty) and this may account for the absent β-strand (β5) and the position of α3 in oxidized Ec-Adx. On the other hand, the missing amide resonances unambiguously identify the presence, and immediate environment, of the paramagnetic [2Fe-2S] cluster in oxidized Ec-Adx (glass half-full).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

Abstract Sequence variation in related proteins is an important characteristic that modulates activity and selectivity. An example of a protein family with a large degree of sequence variation is that of bacterial sortases, which are cysteine transpeptidases on the surface of gram‐positive bacteria. Class A sortases are responsible for attachment of diverse proteins to the cell wall to facilitate environmental adaption and interaction. These enzymes are also used in protein engineering applications for sortase‐mediated ligations (SML) or sortagging of protein targets. We previously investigated SrtA from Streptococcus pneumoniae , identifying a number of putative β7–β8 loop‐mediated interactions that affected in vitro enzyme function. We identified residues that contributed to the ability of S. pneumoniae SrtA to recognize several amino acids at the P1′ position of the substrate motif, underlined in LPXT G , in contrast to the strict P1′ Gly recognition of SrtA from Staphylococcus aureus . However, motivated by the lack of a structural model for the active, monomeric form of S. pneumoniae SrtA, here, we expanded our studies to other Streptococcus SrtA proteins. We solved the first monomeric structure of S. agalactiae SrtA which includes the C‐terminus, and three others of β7–β8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified β7–β8 loop‐mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. A greater understanding of individual SrtA sequence and structural determinants of target selectivity may also facilitate the design or discovery of improved sortagging tools.

Gao, Melody↗
DOE OSTI2023

Unusual modifications of protein biomarkers expressed by plasmid, prophage, and bacterial host of pathogenic Escherichia coli identified using top‐down proteomic analysis

Rationale Pathogenic bacteria often carry prophage (bacterial viruses) and plasmids (small circular pieces of DNA) that may harbor toxin, antibacterial, and antibiotic resistance genes. Proteomic characterization of pathogenic bacteria should include the identification of host proteins and proteins produced by prophage and plasmid genomes. Methods Protein biomarkers of two strains of Shiga toxin–producingEscherichia coli(STEC) were identified using antibiotic induction, matrix‐assisted laser desorption/ionization tandem time‐of‐flight (MALDI‐TOF‐TOF) tandem mass spectrometry (MS/MS) with post‐source decay (PSD), top‐down proteomic (TDP) analysis, and plasmid sequencing. Alphafold2 was also used to compare predicted in silico structures of the identified proteins to prominent fragment ions generated using MS/MS‐PSD. Strain samples were also analyzed with and without chemical reduction treatment to detect the attachment of pendant groups bound by thioester or disulfide bonds. Results Shiga toxin was detected and/or identified in both STEC strains. For the first time, we also identified the osmotically inducible protein (OsmY) whose sequence unexpectedly had two forms: a full and a truncated sequence. The truncated OsmY terminates in the middle of an α‐helix as determined by Alphafold2. A plasmid‐encoded colicin immunity protein was also identified with and without attachment of an unidentified cysteine‐bound pendant group (~307 Da). Plasmid sequencing confirmed top‐down analysis and the identification of a promoter upstream of the immunity gene that is activated by antibiotic induction, that is, SOS box. Conclusions TDP analysis, coupled with other techniques (e.g., antibiotic induction, chemical reduction, plasmid sequencing, and in silico protein modeling), is a powerful tool to identify proteins (and their modifications), including prophage‐ and plasmid‐encoded proteins, produced by pathogenic microorganisms.

Biochemistry & Molecular Biology↗
DOE OSTI2023

A Membrane-Associated Light-Harvesting Model is Enabled by Functionalized Assemblies of Gene-Doubled TMV Proteins

Photosynthetic light harvesting requires efficient energy transfer within dynamic networks of light-harvesting complexes embedded within phospholipid membranes. Artificial light-harvesting models are valuable tools for understanding the structural features underpinning energy absorption and transfer within chromophore arrays. Here, a method for attaching a protein-based light-harvesting model to a planar, fluid supported lipid bilayer (SLB) is developed. The protein model consists of the tobacco mosaic viral capsid proteins that are gene-doubled to create a tandem dimer (dTMV). Assemblies of dTMV break the facial symmetry of the double disk to allow for differentiation between the disk faces. A single reactive lysine residue is incorporated into the dTMV assemblies for the site-selective attachment of chromophores for light absorption. Further, on the opposing dTMV face, a cysteine residue is incorporated for the bioconjugation of a peptide containing a polyhistidine tag for association with SLBs. The dual-modified dTMV complexes show significant association with SLBs and exhibit mobility on the bilayer. The techniques used herein offer a new method for protein-surface attachment and provide a platform for evaluating excited state energy transfer events in a dynamic, fully synthetic artificial light-harvesting system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Rational inhibitor design for Pseudomonas aeruginosa salicylate adenylation enzyme PchD

Pseudomonas aeruginosa is an increasingly antibiotic-resistant pathogen that causes severe lung infections, burn wound infections, and diabetic foot infections. P. aeruginosaproduces the siderophore pyochelin through the use of a non-ribosomal peptide synthetase (NRPS) biosynthetic pathway. Targeting members of siderophore NRPS proteins is one avenue currently under investigation for the development of new antibiotics against antibiotic-resistant organisms. Here, the crystal structure of the pyochelin adenylation domain PchD is reported. The structure was solved to 2.11 Å when co-crystallized with the adenylation inhibitor 5'-O-(N-salicylsulfamoyl)adenosine (salicyl-AMS) and to 1.69 Å with a modified version of salicyl-AMS designed to target an active site cysteine (4-cyano-salicyl-AMS). In the structures, PchD adopts the adenylation conformation, similar to that reported for AB3403 from Acinetobacter baumannii.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Geometry, spin coupling, and dielectric control of redox potentials in [4Fe–4S] Clusters

Iron–sulfur (Fe–S) clusters are common biological cofactors that facilitate vital redox reactions. Despite extensive research, the molecular basis of redox potential tuning in ferredoxin-like proteins remains an active area of debate. In this study, we combine statistical analysis of over one thousand [4Fe–4S]-containing protein structures from the Protein Data Bank (PDB) with broken-symmetry and extended broken-symmetry density functional theory to examine how cysteine ligand orientations and environmental screening affect redox properties of the clusters. We identified five main ligand configurations, three of which are predominant in natural structures. Among these, the adiabatic electron affinity differs by less than 0.1 V, indicating that, while geometry plays a secondary role, it allows localized fine-tuning of redox properties. In contrast, electrostatic and solvation effects primarily determine the overall potential range.

Computational Chemistry↗
DOE OSTI2026

Effect of glutathione-coated Mn-doped ZnS quantum dots on nutrient delivery in basil ( Ocimum basilicum ) plants

Ensuring efficient nutrient delivery while minimizing environmental impacts remains a significant challenge for modern agriculture. Nanotechnology-based fertilizers offer promising strategies to improve nutrient uptake and bioavailability in plants. This research aims to evaluate the use of Glutathione-coated Manganese-doped Zinc Sulfide quantum dots (GSH-ZnS-Mn QDs) as a potential nano fertilizer for basil (Ocimum basilicum). QDs' physicochemical properties were characterized using UV–Vis spectroscopy, photoluminescence, FTIR, and energy-dispersive X-ray spectroscopy, confirming successful Mn doping and glutathione surface functionalization. Basil plants were exposed to different concentrations of GSH-ZnS-Mn QDs under soil and hydroponic conditions. Plant growth parameters, oxidative stress responses, photosynthetic pigments, and macro- and micronutrient uptake were assessed using biochemical assays and inductively coupled plasma optical emission spectrometry (ICP-OES). Elemental uptake, spatial distribution, and zinc speciation were further investigated using synchrotron-based micro-X-ray fluorescence (μ-XRF) imaging and X-ray absorption near-edge structure (XANES) spectroscopy. Results show that exposure to GSH-ZnS-Mn QDs resulted in a concentration-dependent increase in leaf and stem biomass, accompanied by enhanced Zn accumulation in plant tissues. Catalase activity decreased across all tested concentrations, suggesting a shift toward glutathione-dependent antioxidant pathways rather than oxidative damage. Chlorophyll levels exhibited moderate reductions at higher concentrations. The higher increase in macronutrient (K, Ca, and Mg) uptake was reported in plants exposed to 200 ppm of QDs. μ-XRF imaging indicated a selective accumulation of Zn in roots and stems, with partial translocation to leaves. XANES analyses revealed that Zn from QDs was mainly converted into organic Zn species, such as Zn-phytate, Zn-acetate, and Zn-cysteine, indicating transformation and complexation within the plant. The findings demonstrate that a glutathione coating on GSH-ZnS-Mn QDs improves biocompatibility and nutrient delivery efficiency. These results highlight the relevance of surface functionalization in regulating nanoparticle fate, transformation, and nutrient bioavailability, supporting the potential application of GSH–ZnS–Mn QDs as modern nano fertilizers.

Basil↗
DOE OSTI2025

Resolving SPARC–HSA binding kinetics with an ultrasensitive photonic sensor based on bound states in the continuum

Secreted protein acidic and rich in cysteine (SPARC) is critical in cell-matrix interactions and tissue remodeling. It influences tumor progression through its affinity for human serum albumin (HSA) - the most abundant plasma protein, which also plays a crucial role in drug delivery. Strong molecular binding leads to a dissociation constant KD in the nanomolar range. Thus, determining KD requires detecting sub-nanomolar concentrations with ultrasensitive methods. This may be crucial for elucidating the nature of SPARC-HSA binding, as their interaction remains a subject of debate. Capturing these interactions accurately requires a platform capable of resolving rapid binding kinetics at extremely low analyte concentrations. In this work, we report on a microfluidics-integrated photonic nanostructure that supports bound states in the continuum (BICs) and is optimized for studying the fast kinetics of high-affinity protein-protein interactions. The unprecedented capability of detecting sub-nanomolar concentrations allows quantifying KD between SPARC and HSA beyond the state of the art. We leverage an all-dielectric photonic crystal slab (PhCS) sustaining two BIC branches arising from gapped Dirac cone dispersion. HSA is covalently immobilized on the PhCS bonded to a PDMS microfluidic chamber. SPARC dissociation is carried out using PBS buffer (pH 7.4), ensuring complete protein release through precise control of the flow rate and continuous spectral monitoring of the BICs. The measured KD=8.2±0.8 nM confirms the strong affinity of SPARC for HSA. This study highlights the potential of BIC-based sensing as a versatile tool for investigating protein interactions. These results also have implications for the optimization of drug delivery systems and cancer treatment strategies.

Albumin↗
DOE OSTI2022

On the surface passivating principle of functional thiol towards efficient and stable perovskite nanocrystal solar cells

Inorganic halide perovskite nanocrystals (PNCs) have demonstrated promising potential for solar cell applications. However, the lability of photoactive CsPbI3 phase under ambient conditions, coupled with considerable amounts of surface defects induced during solidification process, have impeded achieving high performances and longevities of the PNC-based solar cells. Post-treatment of the PNCs with organic ligands has been proposed as an efficient strategy for surface passivation, which, however, still relies on the binding actions of typical functional groups towards surface defects (especially, carboxylates onto iodine vacancies). Herein, we uncover that thiolate, a deprotonated form of thiol, renders distinctive binding feasibility towards iodine vacancies at the CsPbI3 PNC surface, compared with those of typical functional groups. By treating the PNC solid with deprotonated cysteine as a ligand, the surface defects are comprehensively passivated. The solar cells with the modified PNC films demonstrate an excellent PCE of 15.5 % and improved device longevity (77 % of initial PCE over 2 months) under ambient conditions. Our work not only elucidates the chemical principles of thiol on the binding with PNC surface, but also corroborates the power of thiolate as a promising strategy to develop high performances and improved longevity of solar cells.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Identification of a Covalent Molecular Inhibitor of Anti-apoptotic BFL-1 by Disulfide Tethering

The BCL-2 family is composed of anti- and pro-apoptotic members that respectively protect or disrupt mitochondrial integrity. Anti-apoptotic overexpression can promote oncogenesis by trapping the BCL-2 homology 3 (BH3) “killer domains” of pro-apoptotic proteins in a surface groove, blocking apoptosis. Groove inhibitors, such as the relatively large BCL-2 drug venetoclax (868 Da), have emerged as cancer therapies. BFL-1 remains an undrugged oncogenic protein and can cause venetoclax resistance. Having identified a unique C55 residue in the BFL-1 groove, we performed a disulfide tethering screen to determine if C55 reactivity could enable smaller molecules to block BFL-1's BH3-binding functionality. We found that a disulfide-bearing N-acetyltryptophan analog (304 Da adduct) effectively targeted BFL-1 C55 and reversed BFL-1-mediated suppression of mitochondrial apoptosis. Structural analyses implicated the conserved leucine-binding pocket of BFL-1 as the interaction site, resulting in conformational remodeling. Thus, therapeutic targeting of BFL-1 may be achievable through the design of small, cysteine-reactive drugs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A multi-pronged evaluation of aldehyde-based tripeptidyl main protease inhibitors as SARS-CoV-2 antivirals

As an essential enzyme of SARS-CoV-2, the COVID-19 pathogen, main protease (M Pro ) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 positions and the N-terminal protection group, we synthesized 18 tripeptidyl M Pro inhibitors that contained also an aldehyde warhead and β-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 position. Systematic characterizations of these inhibitors were conducted, including their in vitro enzymatic inhibition potency, X-ray crystal structures of their complexes with M Pro , their inhibition of M Pro transiently expressed in 293T cells, and cellular toxicity and SARS-CoV-2 antiviral potency of selected inhibitors. These inhibitors have a large variation of determined in vitro enzymatic inhibition IC 50 values that range from 4.8 to 650 nM. Here, the determined in vitro enzymatic inhibition IC 50 values reveal that relatively small side chains at both P2 and P3 positions are favorable for achieving high in vitro M Pro inhibition potency, the P3 position is tolerable toward unnatural amino acids with two alkyl substituents on the α-carbon, and the inhibition potency is sensitive toward the N-terminal protection group. X-ray crystal structures of M Pro bound with 16 inhibitors were determined. In all structures, the M Pro active site cysteine interacts covalently with the aldehyde warhead of the bound inhibitor to form a hemithioacetal that takes an S configuration. For all inhibitors, election density around the N-terminal protection group is weak indicating possible flexible binding of this group to M Pro . In M Pro , large structural variations were observed on residues N142 and Q189. Unlike their high in vitro enzymatic inhibition potency, most inhibitors showed low potency to inhibit M Pro that was transiently expressed in 293T cells. Inhibitors that showed high potency to inhibit M Pro transiently expressed in 293T cells all contain O-tert-butyl-threonine at the P3 position. These inhibitors also exhibited relatively low cytotoxicity and high antiviral potency. Overall, our current and previous studies indicate that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for tripeptidyl aldehyde inhibitors of M Pro .

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

A systematic exploration of boceprevir-based main protease inhibitors as SARS-CoV-2 antivirals

Boceprevir is an HCV NSP3 inhibitor that was explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (M Pro ) and contains an α-ketoamide warhead, a P1 β-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert-butylglycine, and a P4 N-terminal tert-butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based M Pro inhibitors including PF-07321332 and characterized their M Pro inhibition potency in test tubes (in vitro) and 293T cells (in cellulo). Crystal structures of M Pro bound with 10 inhibitors and cytotoxicity and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a β-(S-2-oxopyrrolidin-3-yl)-alanyl (Opal) residue and the warhead with an aldehyde leads to high in vitro potency. The original moieties at P2, P3 and the P4 N-terminal cap positions in boceprevir are better than other tested chemical moieties for high in vitro potency. In crystal structures, all inhibitors form a covalent adduct with the M Pro active site cysteine. Here, the P1 Opal residue, P2 dimethylcyclopropylproline and P4 N-terminal tert-butylcarbamide make strong hydrophobic interactions with M Pro , explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains a P4 N-terminal isovaleramide. In its M Pro complex structure, the P4 N-terminal isovaleramide is tucked deep in a small pocket of M Pro that originally recognizes a P4 alanine side chain in a substrate. Although all inhibitors show high in vitro potency, they have drastically different in cellulo potency to inhibit ectopically expressed M Pro in human 293T cells. In general, inhibitors with a P4 N-terminal carbamide or amide have low in cellulo potency. This trend is reversed when the P4 N-terminal cap is changed to a carbamate. The installation of a P3 O-tert-butyl-threonine improves in cellulo potency. Three molecules that contain a P4 N-terminal carbamate were advanced to cytotoxicity tests on 293T cells and antiviral potency tests on three SARS-CoV-2 variants. They all have relatively low cytotoxicity and high antiviral potency with EC 50 values around 1 μM. A control compound with a nitrile warhead and a P4 N-terminal amide has undetectable antiviral potency. Based on all observations, we conclude that a P4 N-terminal carbamate in a boceprevir derivative is key for high antiviral potency against SARS-CoV-2.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Structure-based design of SARS-CoV-2 papain-like protease inhibitors

The COVID-19 pandemic is caused by SARS-CoV-2, an RNA virus with high transmissibility and mutation rate. Given the paucity of orally bioavailable antiviral drugs to combat SARS-CoV-2 infection, there is a critical need for additional antivirals with alternative mechanisms of action. Papain-like protease (PL pro ) is one of the two SARS-CoV-2 encoded viral cysteine proteases essential for viral replication. PL pro cleaves at three sites of the viral polyproteins. In addition, PLpro antagonizes the host immune response upon viral infection by cleaving ISG15 and ubiquitin from host proteins. Therefore, PL pro is a validated antiviral drug target. In this study, we report the X-ray crystal structures of papain-like protease (PL pro ) with two potent inhibitors, Jun9722 and Jun9843 . Subsequently, we designed and synthesized several series of analogs to explore the structure-activity relationship, which led to the discovery of PL pro inhibitors with potent enzymatic inhibitory activity and antiviral activity against SARS-CoV-2. Together, the lead compounds are promising drug candidates for further development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

A deep redox proteome profiling workflow and its application to skeletal muscle of a Duchenne Muscular Dystrophy model

Perturbation to the redox state accompanies many diseases and its effects are viewed through oxidation of biomolecules, including proteins, lipids, and nucleic acids. The thiol groups of protein cysteine residues undergo an array of redox post-translational modifications (PTMs) that are important for regulation of protein and pathway function. To better understand what proteins are redox regulated following a perturbation, it is important to be able to comprehensively profile protein thiol oxidation at the proteome level. Herein, we report a deep redox proteome profiling workflow and demonstrate its application in measuring the changes in thiol oxidation along with global protein expression in skeletal muscle from mdx mice, a model of Duchenne Muscular Dystrophy (DMD). In depth coverage of the thiol proteome was achieved with >18,000 Cys sites from 5608 proteins in muscle being quantified. Compared to the control group, mdx mice exhibit markedly increased thiol oxidation, where ~2% shift in the median oxidation occupancy was observed. Further, pathway analysis for the redox data revealed that coagulation system and immune-related pathways were among the most susceptible to increased thiol oxidation in mdx mice, whereas protein abundance changes were more enriched in pathways associated with bioenergetics. This study illustrates the importance of deep redox profiling in gaining greater insight into oxidative stress regulation and pathways/processes that are perturbed in an oxidizing environment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

High methylation potential of mercury complexed with mixed thiolate ligands by Geobacter sulfurreducens PCA

We report some thiols, such as cysteine (CYS) at moderate concentrations (10–500 µM), can enhance methylmercury (MeHg) formation by Geobacter sulfurreducens PCA, whereas others such as dithiol 2,3-dimercaptopropanesulfonate (DMPS) and 2,3-dimercaptosuccinic acid (DMSA) abolish mercury [Hg(II)] methylation. Little is known, however, about whether Hg(II) methylation could be enhanced or inhibited by the presence of mixed thiol ligands at low concentrations observed in the environment. Surprisingly we found that mixing CYS (1 µM) with DMPS (0.025–0.5 µM) or DMSA (0.025–1 µM) substantially increased MeHg production by 1.5–3.5-fold, compared to the no-thiol control, whereas complexation with a single DMPS, or DMSA, or CYS (1 µM) strongly inhibited Hg(II) methylation. Pre-equilibration between Hg(II) and thiols before the addition of cells was necessary to observe enhanced methylation. Spectroscopic analyses indicated the formation of mixed or heteroleptic coordinated Hg(II)-S 3 /S 4 complexes, which likely facilitated exchange of Hg(II) with cells and its uptake and internal transfer to the HgcAB proteins required for methylation. These results suggest that the effects of thiols on Hg(II) methylation were more complex than previously thought (using a single thiol) and thus underscore the importance of understanding how mixed thiols and their interactions with Hg(II) may ultimately influence MeHg production in the natural environment.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Multi-omics of NET formation and correlations with CNDP1, PSPB, and L-cystine levels in severe and mild COVID-19 infections

We report that we performed a multi-omics analysis of an immunologically naïve SARS-CoV-2 clinical cohort to characterize overall changes in plasma among control (uninfected), mild, and severe infections. A comparison of healthy controls and patient samples showed activation of neutrophil degranulation pathways. Consistent with this observation, we characterized neutrophil extracellular trap (NET) complexes that were partially initiated in a subset of the mild infections (showing partially formed NETs) and fully-formed NETs in a subset of severe infections (containing multiple NET proteins in individual patient samples). As a potential mechanism to suppress NET formation, multiple redox enzymes were elevated in the mild and severe population. Analysis of metabolites from the same cohort showed a 24 and 60-fold elevation in plasma L-cystine, the oxidized form of cysteine and substrate of the powerful antioxidant glutathione in mild and severe patients, respectively. Unique to patients with mild infections, the carnosine dipeptidase modifying enzyme (CNDP1) was up-regulated. The strong protein and metabolite oxidation signatures suggest multiple compensatory pathways working to suppress both free radical and NET formation in SARS-CoV-2 infections.

60 APPLIED LIFE SCIENCES↗