The cross-linking of tyrosine residues in apo-ovotransferrin by treatment with periodate anions.
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ABSTRACT:
SUBMITTER: Hsuan JJ
PROVIDER: S-EPMC1148431 | biostudies-other | 1987 Oct
REPOSITORIES: biostudies-other
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Project description:The reaction between periodate anions and apo-ovotransferrin results in the rapid abolition of the iron-binding ability of the protein and the loss of approximately 4 mol of tyrosine/mol of protein. The degree of inhibition exerted by a variety of salts on the rate of this reaction is found to be inconsistent with the lyotropic series and suggests the existence of a complex anion-binding site in the apoprotein. The existence of this site may explain the action of periodate anions on ovotransferrin.
| S-EPMC1147225 | biostudies-other
Project description:Chemical cross-linking is an attractive approach to map peptide-protein and protein-protein complexes. Previously, we explored 3,4-dihydroxylphenylalanine (DOPA) as a protein cross-linking agent upon periodate oxidation (Burdine, L.; Gillette, T. G.; Lin, H.-J.; Kodadek, T. J. Am. Chem. Soc. 2004, 126, 11442-11443). We report here a study on the chemistry of DOPA-protein cross-linking. First, using a peptide nucleic acid templated system, we identified the alpha-amino, epsilon-amino of Lys, imidazole of His, and thiol of Cys as functional groups capable of attacking DOPA ortho-quinone. Second, we demonstrated that periodate-induced DOPA-protein cross-linking could be carried out efficiently at neutral pH in the presence of excess aliphatic 1,2-diols such as ethylene glycol, lactose, and adenosine triphosphate. This result indicated that DOPA-protein cross-linking and 1,2-diol oxidative cleavage proceed via different mechanisms and that carbohydrates will not interfere with this process when carried out in crude cell extracts or on intact cells.
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Project description:1. Tyrosine was treated with tetranitromethane. 2. Approx. 10% of the tyrosine was converted into 3-nitrotyrosine. 3. Three fluorescent compounds were also formed. They appear to be a dimer, trimer and tetramer in which tyrosine units are linked by biphenyl bonds. 4. The dimer and trimer have also been isolated from some proteins after treatment with tetranitromethane. 5. The yield of 3-nitrotyrosine from ovotransferrin after treatment with tetranitromethane was much smaller than the loss of tyrosine. 6. Several unidentified compounds were also formed by the reaction between tyrosine and tetranitromethane.
| S-EPMC1176556 | biostudies-other
Project description:Tetranitromethane reacts with essentially all 21 tyrosine residues of iron-free ovotransferrin. In iron-ovotransferrin, 7 mol of tyrosine/mol of protein are unreactive. Peptides containing the unreactive tyrosine residues were isolated from digests of nitrated iron-ovotransferrin. By comparing the structures of the peptides with the amino acid sequence of ovotransferrin it is found that there are ten protected residues occupying positions 42, 82, 92, 188, 319, 415, 431, 521 and 524 in the polypeptide chain. The problem of identifying the tyrosine residues that form bonds with the metal atoms is discussed.
| S-EPMC1163693 | biostudies-other
Project description:Treatment of isolated human erythrocyte membranes at pH 7.4 with 0.1-0.5 mM-sodium periodate specifically cross-linked some of the spectrin polypeptides. Treatment with 2 mM-periodate resulted in complete cross-linking of spectrin and partial cross-linking of other polypeptides. The latter treatment also caused aggregation of the intramembrane particles made visible by freeze-fracturing. When membranes that had been treated with 2 mM-periodate were depleted of spectrin by treatment with 0.1 mM-EDTA, extensive aggregation of the intramembrane particles occurred.
| S-EPMC1184014 | biostudies-other
Project description:The gas-phase oxidation of doubly protonated peptides containing neutral basic residues to various products, including [M + H + O]+, [M - H]+, and [M - H - NH3]+, is demonstrated here via ion/ion reactions with periodate. It was previously demonstrated that periodate anions are capable of oxidizing disulfide bonds and methionine, tryptophan, and S-alkyl cysteine residues. However, in the absence of these easily oxidized sites, we show here that systems containing neutral basic residues can undergo oxidation. Furthermore, we show that these neutral basic residues primarily undergo different types of oxidation (e.g., hydrogen abstraction) reactions than those observed previously (i.e., oxygen transfer to yield the [M + H + O]+ species) upon gas-phase ion/ion reactions with periodate anions. This chemistry is illustrated with a variety of systems, including a series of model peptides, a cell-penetrating peptide containing a large number of unprotonated basic sites, and ubiquitin, a roughly 8.6 kDa protein. Graphical Abstract ᅟ.
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Project description:Engineered protein hydrogels have shown promise as artificial extracellular matrix materials for the 3D culture of stem cells due to the ability to decouple hydrogel biochemistry and mechanics. The modular design of these proteins allows for incorporation of various bioactive sequences to regulate cellular behavior. However, the chemistry used to cross-link the proteins into hydrogels can limit what bioactive sequences can be incorporated, in order to prevent nonspecific cross-linking within the bioactive region. Bio-orthogonal cross-linking chemistries may allow for the incorporation of any arbitrary bioactive sequence, but site-selective and scalable incorporation of bio-orthogonal reactive groups such as azides that do not rely on commonly used amine-reactive chemistry is often challenging. In response, we have optimized the reaction of an azide-bearing 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) with engineered elastin-like proteins (ELPs) to selectively azide-functionalize tyrosine residues within the proteins. The PTAD-azide functionalized ELPs cross-link with bicyclononyne (BCN) functionalized ELPs via the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction to form hydrogels. Human mesenchymal stem cells and murine neural progenitor cells encapsulated within these hydrogels remain highly viable and maintain their phenotypes in culture. Tyrosine-specific modification may expand the number of bioactive sequences that can be designed into protein-engineered materials by permitting incorporation of lysine-containing sequences without concern for nonspecific cross-linking.
| S-EPMC7274545 | biostudies-literature
Project description:Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. This work describes the development of peptide activity/affinity-based probes for the discovery of proteins that recognize novel acyl-based PTMs on lysine residues in the proteome. The probes contain surrogates of ?-N-acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the experiments. In addition to the modified PTMs, the developed chemotypes were analyzed with respect to the effect of peptide sequence. The photo cross-linking conditions and subsequent functionalization of the covalent adducts were systematically optimized by applying fluorophore labeling and gel electrophoresis (in-gel fluorescence measurements). Finally, selected probes, containing the ?-N-glutaryllysine and ?-N-myristoyllysine analogues, were successfully applied for the enrichment of native, endogenous proteins from cell lysate, recapitulating the expected interactions of SIRT5 and SIRT2, respectively. Interestingly, the latter mentioned was able to pull down two different splice variants of SIRT2, which has not been achieved with a covalent probe before. Based on this elaborate proof-of-concept study, we expect that the technology will have broad future applications for pairing of novel PTMs with the proteins that target them in the cell.
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Project description:The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches.
| S-EPMC4084482 | biostudies-literature
Project description:Viral capsid dynamics are often observed during infectious events such as cell surface attachment, entry and genome release. Structural analysis of adeno-associated virus (AAV), a helper-dependent parvovirus, revealed a cluster of surface-exposed tyrosine residues at the icosahedral two-fold symmetry axis. We exploited the latter observation to carry out selective oxidation of Tyr residues, which yielded cross-linked viral protein (VP) subunit dimers, effectively "stitching" together the AAV capsid two-fold interface. Characterization of different Tyr-to-Phe mutants confirmed that the formation of cross-linked VP dimers is mediated by dityrosine adducts and requires the Tyr704 residue, which crosses over from one neighboring VP subunit to the other. When compared to unmodified capsids, Tyr-cross-linked AAV displayed decreased transduction efficiency in cell culture. Surprisingly, further biochemical and quantitative microscopy studies revealed that restraining the two-fold interface hinders externalization of buried VP N-termini, which contain a phospholipase A2 domain and nuclear localization sequences critical for infection. These adverse effects caused by tyrosine oxidation support the notion that interfacial dynamics at the AAV capsid two-fold symmetry axis play a role in externalization of VP N-termini during infection.
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