Selenium Decipher: Trapping of Native Selenomethionine-Containing Peptides in Selenium-Enriched Milk and Unveiling the Deterioration after Ultrahigh-Temperature Treatment.

Selenopeptide identification relies on databases to interpret the selenopeptide spectra. A common database search strategy is to set selenium as a variable modification instead of sulfur on peptides. However, this approach generally detects only a fraction of selenopeptides. An alternative approach, termed Selenium Decipher, is proposed in the present study. It involves identifying collision-induced dissociation-cleavable selenomethionine-containing peptides by iteratively matching the masses of seleno-amino acids in selenopeptide spectra. This approach uses variable-data-independent acquisition (vDIA) for peptide detection, providing a flexible and customizable window for secondary mass spectral fragmentation. The attention mechanism was used to capture global information on peptides and determine selenomethionine-containing peptide backbones. The core structure of selenium on selenomethionine-containing peptides generates a series of fragment ions, namely, C3H7Se+, C4H10NSe+, C5H7OSe+, C5H8NOSe+, and C7H11N2O2Se+, with known mass gaps during higher-energy collisional dissociation (HCD) fragmentation. De-selenium spectra are generated by removing selenium originating from selenium replacement and then reassigning the precursors to peptides. Selenium-enriched milk is obtained by feeding selenium-rich forage fed to cattle, which leads to the formation of native selenium through biotransformation. A novel antihypertensive selenopeptide Thr-Asp-Asp-Ile-SeMet-Cys-Val-Lys TDDI(Se)MCVK was identified from selenium-enriched milk. The selenopeptide (IC50 = 60.71 µM) is bound to four active residues of the angiotensin-converting enzyme (ACE) active pocket (Ala354, Tyr523, His353, and His513) and two active residues of zinc ligand (His387 and Glu411) and exerted a competitive inhibitory effect on the spatial blocking of active sites. The integration of vDIA and the iteratively matched seleno-amino acids was applied for Selenium Decipher, which provides high validity for selenomethionine-containing peptide identification.

82Se Metabolically-Labeled Yeast as a Matrix-Matched Isotope Dilution Standard for Quantification of Selenomethionine.

Selenized yeast is commonly used as a highly bioavailable source of selenium in dietary supplements and feed additives and is used in research settings in various disciplines due to the large number of selenium-containing metabolites formed during growth. With the selenomethionine being the major form of selenium present in selenized yeasts, its accurate quantitation is essential, however, values are frequently underestimated due to the costly and time-consuming hydrolysis-based sample preparation required to release the selenoamino acid from proteins for analysis. The National Research Council Canada has developed an 82-Se-enriched selenized yeast Certified Reference Material, SEEY-1 (DOI: 10.4224/crm.2023.seey-1) intended to be used as a matrix-matched spike material for isotope dilution analysis of selenized yeasts. The total selenium and selenomethionine contents of SEEY-1 were determined to be 322.1 ± 4.8 mg/kg (k = 2) and 635.6 ± 16.8 mg/kg (k = 2), respectively. Here we present results on the preparation of the 82-Se-enriched yeast, the certification process, and provide an example of the use of SEEY-1 as a matrix-matched spike for the analysis of selenomethionine in a sample of selenized yeast. We demonstrate here that SEEY-1 is able to compensate for the partial digestion of yeast proteins and provide reliable analytical data on Se amino acid content in under an hour instead of the 16 hours required for conventional complete acid hydrolysis.

An analysis of the content changes in free and combinative forms of organic selenium in radish sprouts cultivated with solutions of selenoamino acids.

Selenium (Se) is critical for human health, but human intake of Se is often inadequate. Organic forms of dietary Se are considered safe and more bioavailable than inorganic forms. Along with a generally high nutritional value, sprouts are sensitive to Se treatment. This study used selenomethionine and methylselenocysteine solutions to cultivate Se-enriched sprouts under an optimized hydroponic condition. Content change and transformations of the selenoamino acids were analyzed by a developed HPLC-ESI-MS/MS method. Uptake of both selenomethionine and methylselenocysteine was dose-dependent and involved active transport and passive diffusion, as demonstrated by the respiratory and aquaporin inhibition assays. Passive diffusion played a dominant role. Free methylselenocysteine was the predominant form in samples. Selenomethionine and methylselenocysteine were capable of mutual transformation. Moreover, the selenoprotein generation was associated with the increasing Se concentration of the culture solutions. The results provided scientific references for the efficient utilization of organic Se in sprouts.

Chalcogen Bonds Involving Selenium in Protein Structures.

Chalcogen bonds are the specific interactions involving group 16 elements as electrophilic sites. The role of chalcogen atoms as sticky sites in biomolecules is underappreciated, and the few available studies have mostly focused on S. Here, we carried out a statistical analysis over 3562 protein structures in the Protein Data Bank (PDB) containing 18 266 selenomethionines and found that Se···O chalcogen bonds are commonplace. These findings may help the future design of functional peptides and contribute to understanding the role of Se in nature.

Charge Assisted S/Se Chalcogen Bonds in SAM Riboswitches: A Combined PDB and ab Initio Study.

In this study, we provide experimental (Protein Data Bank (PDB) inspection) and theoretical (RI-MP2/def2-TZVP level of theory) evidence of the involvement of charge assisted chalcogen bonding (ChB) interactions in the recognition and folding mechanisms of S-adenosylmethionine (SAM) riboswitches. Concretely, an initial PDB search revealed several examples where ChBs between S-adenosyl methionine (SAM)/adenosyl selenomethionine (EEM) molecules and uracil (U) bases belonging to RNA take place. While these interactions are usually described as a merely Coulombic attraction between the positively charged S/Se group and RNA, theoretical calculations indicated that the σ holes of S and Se are involved. Moreover, computational models shed light on the strength and directionality properties of the interaction, which was also further characterized from a charge-density perspective using Bader's "Atoms in Molecules" (AIM) theory, Non-Covalent Interaction plot (NCIplot) visual index, and Natural Bonding Orbital (NBO) analyses. As far as our knowledge extends, this is the first time that ChBs in SAM-RNA complexes have been systematically analyzed, and we believe the results might be useful for scientists working in the field of RNA engineering and chemical biology as well as to increase the visibility of the interaction among the biological community.

Analytical Problems in Separation of Selenomethionine and Its Oxidative Product in HILIC HPLC.

Selenomethionine (SeMet) is one of the main selenium forms in foods and supplements. Determining its presence in natural food samples creates difficulties due to possible oxidation processes. The objective of this study was to evaluate the possible degradation of SeMet in water extracts of green teas, one of the most consumed beverages worldwide. Such a medium has not been investigated at this time. The HILIC-HPLC MS/MS method with different stationary phases was used to achieve the satisfactory separation of SeMet and selenomethionine oxide (SeMetO). The addition of dithiothreitol and ß-mercaptoethanol, recommended to ensure that SeMet is kept in the reduced form, was also evaluated. The best separation was achieved using the zwitterionic HILIC stationary phase coupled to mass spectrometry and MeOH with water (85/15, v/v) as the eluent. Extraction was done with hot water with the addition of ß-mercaptoethanol. The infusions prepared from Lung-Ching teas (from the Zhejiang Province in China) contained the highest concentration of selenium in a typical cup of tea (12.5-17.3 µg L-1). For other tested teas it decreased in the following order: Yunnan > Dilmah > Lipton. For Lung-Ching teas, the sum of concentrations of SeMet and SeMetO corresponded to about 46-63% of the total selenium in their extracts.

Structural Characterization of a Single-Stranded DNA-Binding Protein: A Case Study of the ORF6 Protein from Bacteriophage Enc34.

In the quest to understand how single-stranded DNA-binding proteins function and evolve at a molecular level, determination of their high-resolution three-dimensional structure using methods such as X-ray crystallography is indispensable. Here we present a collection of methods used in crystallographic studies of the single-stranded DNA-binding protein from the bacteriophage Enc34, from designing expression constructs through to protein production, purification, and crystallization, to determination and analysis of the protein's three-dimensional structure. The chapter aims to shed light on all the essential stages in a structural study of a single-stranded DNA-binding protein, with a spotlight on procedures specific to X-ray crystallography to aid those interested in venturing into structural biology.

Selenium Biofortification: Roles, Mechanisms, Responses and Prospects.

The trace element selenium (Se) is a crucial element for many living organisms, including soil microorganisms, plants and animals, including humans. Generally, in Nature Se is taken up in the living cells of microorganisms, plants, animals and humans in several inorganic forms such as selenate, selenite, elemental Se and selenide. These forms are converted to organic forms by biological process, mostly as the two selenoamino acids selenocysteine (SeCys) and selenomethionine (SeMet). The biological systems of plants, animals and humans can fix these amino acids into Se-containing proteins by a modest replacement of methionine with SeMet. While the form SeCys is usually present in the active site of enzymes, which is essential for catalytic activity. Within human cells, organic forms of Se are significant for the accurate functioning of the immune and reproductive systems, the thyroid and the brain, and to enzyme activity within cells. Humans ingest Se through plant and animal foods rich in the element. The concentration of Se in foodstuffs depends on the presence of available forms of Se in soils and its uptake and accumulation by plants and herbivorous animals. Therefore, improving the availability of Se to plants is, therefore, a potential pathway to overcoming human Se deficiencies. Among these prospective pathways, the Se-biofortification of plants has already been established as a pioneering approach for producing Se-enriched agricultural products. To achieve this desirable aim of Se-biofortification, molecular breeding and genetic engineering in combination with novel agronomic and edaphic management approaches should be combined. This current review summarizes the roles, responses, prospects and mechanisms of Se in human nutrition. It also elaborates how biofortification is a plausible approach to resolving Se-deficiency in humans and other animals.

Selenomethionine as an expressible handle for bioconjugations.

Site-selective chemical bioconjugation reactions are enabling tools for the chemical biologist. Guided by a careful study of the selenomethionine (SeM) benzylation, we have refined the reaction to meet the requirements of practical protein bioconjugation. SeM is readily introduced through auxotrophic expression and exhibits unique nucleophilic properties that allow it to be selectively modified even in the presence of cysteine. The resulting benzylselenonium adduct is stable at physiological pH, is selectively labile to glutathione, and embodies a broadly tunable cleavage profile. Specifically, a 4-bromomethylphenylacetyl (BrMePAA) linker has been applied for efficient conjugation of complex organic molecules to SeM-containing proteins. This expansion of the bioconjugation toolkit has broad potential in the development of chemically enhanced proteins.

Expression, purification and crystallization of the N-terminal Solanaceae domain of the Sw-5b NLR immune receptor.

Plant nucleotide-binding domain and leucine-rich repeat receptors (NLRs) play crucial roles in recognizing pathogen effectors and activating plant immunity. The tomato NLR Sw-5b is a coiled-coil NLR (CC-NLR) immune receptor that confers resistance against tospoviruses, which cause serious economic losses in agronomic crops worldwide. Compared with other CC-NLRs, Sw-5b possesses an extended N-terminal Solanaceae domain (SD). The SD of Sw-5b is critical for recognition of the tospovirus viral movement protein NSm. An SD is also frequently detected in many NLRs from Solanaceae plants. However, no sequences homologous to the SD have been detected in animals or in plants other than Solanaceae. The properties of the SD protein are largely unknown, and thus 3D structural information is vital in order to better understand its role in pathogen perception and the activation of immune receptors. Here, the expression, purification and crystallization of Sw-5b SD (amino acids 1-245) are reported. Native and selenomethionine-substituted crystals of the SD protein belonged to space group P3112, with unit-cell parameters a = 81.53, b = 81.53, c = 98.44 Šand a = 81.63, b = 81.63, c = 98.80 Å, respectively. This is the first report of a structural study of the noncanonical SD domain of the NLR proteins from Solanaceae plants.

77Se NMR Probes the Protein Environment of Selenomethionine.

Sulfur is critical for the correct structure and proper function of proteins. Yet, lacking a sensitive enough isotope, nuclear magnetic resonance (NMR) experiments are unable to deliver for sulfur in proteins the usual wealth of chemical, dynamic, and structural information. This limitation can be circumvented by substituting sulfur with selenium, which has similar physicochemical properties and minimal impact on protein structures but possesses an NMR compatible isotope (77Se). Here we exploit the sensitivity of 77Se NMR to the nucleus' chemical milieu and use selenomethionine as a probe for its proteinaceous environment. However, such selenium NMR spectra of proteins currently resist a reliable interpretation because systematic connections between variations of system variables and changes in 77Se NMR parameters are still lacking. To start narrowing this knowledge gap, we report here on a biological 77Se magnetic resonance data bank based on a systematically designed library of GB1 variants in which a single selenomethionine was introduced at different locations within the protein. We recorded the resulting isotropic 77Se chemical shifts and relaxation times for six GB1 variants by solution-state 77Se NMR. For four of the GB1 variants we were also able to determine the chemical shift anisotropy tensor of SeM by solid-state 77Se NMR. To enable interpretation of the NMR data, the structures of five of the GB1 variants were solved by X-ray crystallography to a resolution of 1.2 Å, allowing us to unambiguously determine the conformation of the selenomethionine. Finally, we combine our solution- and solid-state NMR data with the structural information to arrive at general insights regarding the execution and interpretation of 77Se NMR experiments that exploit selenomethionine to probe proteins.

Structure determination of the nucleosome core particle by selenium SAD phasing.

The eukaryotic genome is compacted inside the nucleus of the cell in the form called chromatin. The fundamental unit of chromatin is the nucleosome, which contains four types of histones (H3, H4, H2A and H2B) and approximately 150 base pairs of DNA wrapped around the histone complex. The structure of the nucleosome is highly conserved across several eukaryotic species, and molecular replacement has been the primary phasing method used to solve nucleosome structures by X-ray crystallography. However, there is currently no simple, widely applicable experimental phasing method for the nucleosome. In the present study, it is demonstrated that selenomethionine-incorporated histones H3, H2A and H2B can be reconstituted into nucleosomes and crystallized for structural determination. Unexpectedly, it was found that the nucleosome can be phased with a relatively small number of Se atoms. The structures of nucleosome core particles containing 12 and 16 Se atoms were solved by SAD phasing at 2.5 and 2.4 Šresolution, respectively. The present study demonstrates a simple method for determining nucleosome structures by experimental phasing, which may be particularly useful for noncanonical structures that cannot be solved by molecular replacement.

Catalytic M Center of Copper Monooxygenases Probed by Rational Design. Effects of Selenomethionine and Histidine Substitution on Structure and Reactivity.

The M centers of the mononuclear monooxygenases peptidylglycine monooxygenase (PHM) and dopamine ß-monooxygenase bind and activate dioxygen en route to substrate hydroxylation. Recently, we reported the rational design of a protein-based model in which the CusF metallochaperone was repurposed via a His to Met mutation to act as a structural and spectroscopic biomimic. The PHM M site exhibits a number of unusual attributes, including a His2Met ligand set, a fluxional Cu(I)-S(Met) bond, tight binding of exogenous ligands CO and N3-, and complete coupling of oxygen reduction to substrate hydroxylation even at extremely low turnover rates. In particular, mutation of the Met ligand to His completely eliminates the catalytic activity despite the propensity of CuI-His3 centers to bind and activate dioxygen in other metalloenzyme systems. Here, we further develop the CusF-based model to explore methionine variants in which Met is replaced by selenomethionine (SeM) and histidine. We examine the effects on coordinate structure and exogenous ligand binding via X-ray absorption spectroscopy and electron paramagnetic resonance and probe the consequences of mutations on redox chemistry via studies of the reduction by ascorbate and oxidation via molecular oxygen. The M-site model is three-coordinate in the Cu(I) state and binds CO to form a four-coordinate carbonyl. In the oxidized forms, the coordination changes to tetragonal five-coordinate with a long axial Met ligand that like the enzymes is undetectable at either the Cu or Se K edges. The EXAFS data at the Se K edge of the SeM variant provide unique information about the nature of the Cu-methionine bond that is likewise weak and fluxional. Kinetic studies document the sluggish reactivity of the Cu(I) complexes with molecular oxygen and rapid rates of reduction of the Cu(II) complexes by ascorbate, indicating a remarkable stability of the Cu(I) state in all three derivatives. The results show little difference between the Met ligand and its SeM and His congeners and suggest that the Met contributes to catalysis in ways that are more complex than simple perturbation of the redox chemistry. Overall, the results stimulate a critical re-examination of the canonical reaction mechanisms of the mononuclear copper monooxygenases.

Complete labelling of pneumococcal DNA-binding proteins with seleno-L-methionine.

Streptococcus pneumoniae is a pathogenic and opportunistic Gram-positive bacterium that is the leading cause of community-acquired respiratory diseases, varying from mild- to deathly- infections. The appearance of antibiotic-resistant isolates has prompted the search for novel strategies and targets to tackle the bacterial resistances. One of the most promising approaches is the structure-based knowledge of possible targets in conjunction with rational design and docking of inhibitors of the chosen targets. A useful technique that helps to solve protein structures is to label them with an amino acid derivative like seleno-methionine that facilitates tracing of some of the amino acid residues. We have chosen two pneumococcal DNA-binding proteins, namely the relaxase domain of MobM protein from plasmid pMV158, and the RelB-RelE antitoxin-toxin protein complex. Through several changes that improve substantially a previous protocol (Budisa et al., 1995), we have used seleno-L-methionine to incorporate selenium into the amino acid sequence of the selected proteins. We have achieved 100% labelling of the proteins and could demonstrate that the labelled proteins retained full activity as judged from the relaxation of supercoiled plasmid DNA and from gel-retardation assays.

Water-Mediated Selenium Hydrogen-Bonding in Proteins: PDB Analysis and Gas-Phase Spectroscopy of Model Complexes.

High-resolution X-ray crystallography and two-dimensional NMR studies demonstrate that water-mediated conventional hydrogen-bonding interactions (N-H···N, O-H···N, etc.) bridging two or more amino acid residues contribute to the stability of proteins and protein-ligand complexes. In this work, we have investigated single water-mediated selenium hydrogen-bonding interactions (unconventional hydrogen-bonding) between amino acid residues in proteins through extensive protein data bank (PDB) analysis coupled with gas-phase spectroscopy and quantum chemical calculation of a model complex consisting of indole, dimethyl selenide, and water. Here, indole and dimethyl selenide represent the amino acid residues tryptophan and selenomethionine, respectively. The current investigation demonstrates that the most stable structure of the model complex observed in the IR spectroscopy mimics single water-mediated selenium hydrogen-bonded structural motifs present in the crystal structures of proteins. The present work establishes that water-mediated Se hydrogen-bonding interactions are ubiquitous in proteins and the number of these interactions observed in the PDB is more than that of direct Se hydrogen-bonds present there.

Methionine and Selenomethionine as Energy Transfer Acceptors for Biomolecular Structure Elucidation in the Gas Phase.

Mass spectrometry affords rapid and sensitive analysis of peptides and proteins. Coupling spectroscopy with mass spectrometry allows for the development of new methods to enhance biomolecular structure determination. Herein, we demonstrate two new energy acceptors that can be utilized for action-excitation energy transfer experiments. In the first system, C-S bonds in methionine act as energy acceptors from native chromophores, including tyrosine, tryptophan, and phenylalanine. Comparison among chromophores reveals that tyrosine transfers energy most efficiently at 266 nm, but phenylalanine and tryptophan also transfer energy with comparable efficiencies. Overall, the C-S bond dissociation yields following energy transfer are low for methionine, which led to an investigation of selenomethionine, a common analog that is found in many naturally occurring proteins. Sulfur and selenium are chemically similar, but C-Se bonds are weaker than C-S bonds and have lower lying σ* anti-bonding orbitals. Excitation of peptides containing tyrosine and tryptophan results in efficient energy transfer to selenomethionine and abundant C-Se bond dissociation. A series of helical peptides were examined where the positions of the donor or acceptor were systematically scanned to explore the influence of distance and helix orientation on energy transfer. The distance was found to be the primary factor affecting energy transfer efficiency, suggesting that selenomethionine may be a useful acceptor for probing protein structure in the gas phase.

A micellar electrokinetic chromatography approach using diastereomeric derivatization and a volatile surfactant for the enantioselective separation of selenomethionine.

A MEKC methodology with UV detection was developed for the enantioselective separation of selenomethionine (SeMet). The use of (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as chiral derivatization reagent to form SeMet diastereomers enabled their subsequent separation using ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase. The effect of APFO concentration and pH, temperature, injection volume, and derivatization conditions (time and FLEC/SeMet ratio) were evaluated in order to select the best separation conditions. A chiral resolution of 4.4 for DL-SeMet was achieved in less than 6 min using 100 mM APFO at pH 8.5 as electrophoretic buffer. Satisfactory results were obtained in terms of linearity, precision (RSD from 3.4 to 5.1% for migration times and from 1.8 to 4.6% for corrected peak areas), accuracy, and LODs (3.1 × 10-6  M and 3.7 × 10-6  M for d and l enantiomers, respectively). The method was successfully applied to the determination of l-SeMet in food supplements.

Isolation and identification of immunomodulatory selenium-containing peptides from selenium-enriched rice protein hydrolysates.

The RAW264.7 cell model was employed to screen immunomodulatory selenium-containing peptides from selenium-enriched rice protein hydrolysates (SPHs). Moreover, the selenium-containing peptides of high-activity protein hydrolysates were purified by Sephadex G-25, and identified by reversed phase ultra performance liquid chromatography coupled with triple quadrupole time-of-flight mass spectrometry. The results showed that 25 peptide sequences containing selenomethionine (SeMet) information above 90% of probability confidence were found in a fraction of alcalase hydrolysates. SeMDPGQQ and TSeMMM of 100% probability confidence were speculated as two novel selenium-containing peptide sequences. The artificially synthesized peptide TSeMMM was subsequently verified by an excellent immunomodulatory activity at a concentration of 80 µg/mL. In conclusion, the immunomodulatory activity of SPHs was correlated to SeMet sequence in the structure of selenium-containing peptides, and TSeMMM with a stronger immunomodulatory activity demonstrated potential as functional food additives for improving human health.

Selenomethionine and Methioninase: Selenium Free Radical Anticancer Activity.

Colloidal selenium, was first used to treat cancer as early as 1911 in both humans and mice. Selenium was identified as the toxic component in forage plants of sheep, cattle, and horses in the 1930s. The animal toxicity of selenium compounds was determined to be from the metabolism by animals of the elevated concentrations of Se-methylselenocysteine and selenomethionine in plants. The metabolism of both Se-methylselenocysteine and selenomethionine by animals gives rise to the metabolite, methylselenide (CH3Se-), which if in sufficient concentration oxidizes thiols and generates superoxide and other reactive oxygen species. Cancer cells that may overly express methionine gamma-lyase, or beta-lyase (methioninase), by induced viral genomic expression, are susceptible to free radical-induced apoptosis from selenomethionine or Se-methylselenocysteine supplementation.

Direct Imaging of Protein-Specific Methylation in Mammalian Cells.

Abundant post-translational modification through methylation alters the function, stability, and/or localization of a protein. Malfunctions in post-translational modification are associated with severe diseases. To unravel protein methylation sites and their biological functions, chemical methylation reporters have been developed. However, until now, their usage was limited to cell lysates. Herein, we present the first generally applicable approach for imaging methylation of individual proteins in human cells, which is based on a combination of chemical reporter strategies, bioorthogonal ligation reactions, and FRET detected by means of fluorescence lifetime imaging microscopy. Through this approach, methylation of histone 4 and the non-histone proteins tumor suppressor p53, kinase Akt1, and transcription factor Foxo1 in two human cell lines has been successfully imaged. To further demonstrate its potential, the localization-dependent methylation state of Foxo1 in the cellular context has been visualized.