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The Biochemical Journal Apr 1987Rat embryo fibroblasts, line 3Y1, were prelabelled for 24 h with [35S]sulphate and incubated in fresh medium without [35S]sulphate. A rapid efflux of the overall...
Rat embryo fibroblasts, line 3Y1, were prelabelled for 24 h with [35S]sulphate and incubated in fresh medium without [35S]sulphate. A rapid efflux of the overall 35S-labelled compounds from the cells into the medium was observed. After 9 h of incubation, about 50% of the total 35S radioactivity appeared in the medium and up to 84.3% did so at the end of a 48 h incubation. Determination of [35S]sulphated macromolecules present in both the cell-associated and the incubation-medium fractions at different time points during incubation indicated that the majority of the 35S-labelled compounds released from the cells were low-Mr products derived from digestion of the [35S]sulphated macromolecules. Further analysis for tyrosine-O-[35S]sulphated proteins, which constituted only a small fraction of the overall [35S]sulphated macromolecules, showed that, after 9 h of incubation, there was a 65% decrease in the cell-associated fraction, and only 16.4% remained after 48 h. During that time, an amount equivalent to 20.7% of the cell-associated tyrosine-O-[35S]sulphated proteins originally present was released into the medium. Free tyrosine O-[35S]sulphate was generated in the cells and excreted into the incubation medium. Its rate of increase with time, however, was slow, and could account for only 12.4% of the tyrosine-O-[35S]sulphated proteins catabolized at the end of the 48 h incubation.
Topics: Animals; Cell Line; Electrophoresis; Fetal Proteins; Fibroblasts; Macromolecular Substances; Rats; Tyrosine
PubMed: 3632633
DOI: 10.1042/bj2430555 -
Biophysical Journal Nov 2020
Topics: Cyclins; Phosphorylation; Proliferating Cell Nuclear Antigen; Signal Transduction; Tyrosine
PubMed: 33120016
DOI: 10.1016/j.bpj.2020.09.036 -
Antioxidants & Redox Signaling Oct 2013The conversion of protein-bound Tyr residues to 3-nitrotyrosine (3NY) can occur during nitrative stress and has been correlated to aging and many disease states.... (Review)
Review
SIGNIFICANCE
The conversion of protein-bound Tyr residues to 3-nitrotyrosine (3NY) can occur during nitrative stress and has been correlated to aging and many disease states. Proteomic analysis of this post-translational modification, using mass spectrometry-based techniques, is crucial for understanding its potential role in pathological and physiological processes.
RECENT ADVANCES
To overcome some of the disadvantages inherent to well-established nitroproteomic methods using anti-3NY antibodies and gel-based separations, methods involving multidimensional chromatography, precursor ion scanning, and/or chemical derivatization have emerged for both identification and quantitation of protein nitration sites. A few of these methods have successfully detected endogenous 3NY modifications from biological samples.
CRITICAL ISSUES
While model systems often show promising results, identification of endogenous 3NY modifications remains largely elusive. The frequently low abundance of nitrated proteins in vivo, even under inflammatory conditions, is especially challenging, and sample loss due to derivatization and cleaning may become significant.
FUTURE DIRECTIONS
Continued efforts to avoid interference from non-nitrated peptides without sacrificing recovery of nitrated peptides are needed. Quantitative methods are emerging and are crucial for identifying endogenous modifications that may have significant biological impacts.
Topics: Animals; Chromatography; Humans; Oxidation-Reduction; Proteins; Proteomics; Tyrosine
PubMed: 23157221
DOI: 10.1089/ars.2012.5058 -
Journal of the American Chemical Society Apr 2018Introducing new chemical reactivity into proteins in living cells would endow innovative covalent bonding ability to proteins for research and engineering in vivo....
Introducing new chemical reactivity into proteins in living cells would endow innovative covalent bonding ability to proteins for research and engineering in vivo. Latent bioreactive unnatural amino acids (Uaas) can be incorporated into proteins to react with target natural amino acid residues via proximity-enabled reactivity. To expand the diversity of proteins amenable to such reactivity in vivo, a chemical functionality that is biocompatible and able to react with multiple natural residues under physiological conditions is highly desirable. Here we report the genetic encoding of fluorosulfate-l-tyrosine (FSY), the first latent bioreactive Uaa that undergoes sulfur-fluoride exchange (SuFEx) on proteins in vivo. FSY was found nontoxic to Escherichia coli and mammalian cells; after being incorporated into proteins, it selectively reacted with proximal lysine, histidine, and tyrosine via SuFEx, generating covalent intraprotein bridge and interprotein cross-link of interacting proteins directly in living cells. The proximity-activatable reactivity, multitargeting ability, and excellent biocompatibility of FSY will be invaluable for covalent manipulation of proteins in vivo. Moreover, genetically encoded FSY hereby empowers general proteins with the next generation of click chemistry, SuFEx, which will afford broad utilities in chemical biology, drug discovery, and biotherapeutics.
Topics: Escherichia coli Proteins; Fluorides; Genetic Code; HEK293 Cells; HeLa Cells; Histidine; Humans; Lysine; Models, Molecular; Sulfur; Sulfuric Acids; Tyrosine
PubMed: 29601199
DOI: 10.1021/jacs.8b01087 -
Biochemistry. Biokhimiia Jun 2019Neutrophil myeloperoxidase (MPO) plays an important role in protecting the body against infections. MPO products - hypohalous acids and phenoxyl radicals - are strong...
Neutrophil myeloperoxidase (MPO) plays an important role in protecting the body against infections. MPO products - hypohalous acids and phenoxyl radicals - are strong oxidants that can damage not only foreign intruders but also host tissues, including blood plasma proteins. Here, we compared the MPO-induced oxidation of two plasma proteins with antioxidant properties - human serum albumin (HSA) and ceruloplasmin (CP). Incubation of both proteins with hypochlorite (NaOCl) or catalytically active MPO (MPO + H2O2), which synthesizes hypochlorous acid (HOCl) in the presence of chloride ions, resulted in the quenching of protein tryptophan fluorescence. Oxidation-induced changes in the structures of HSA and CP were different. HSA efficiently neutralized MPO-generated oxidants without protein aggregation, while CP oxidation resulted in the formation of large aggregates stabilized by strong covalent bonds between the aromatic amino acid residues. Tyrosine is present in the plasma as free amino acid and also as a component of the polypeptide chains of the proteins. The number of tyrosine residues in a protein does not determine its propensity for aggregate formation. In the case of CP, protein aggregation was primarily due to the high content of tryptophan residues in its polypeptide chain. MPO-dependent oxidation of free tyrosine results in the formation of tyrosyl radicals, that do not oxidize aromatic amino acid residues in proteins because of the high rate of recombination with dityrosine formation. At the same time, free tyrosine can influence MPO-induced protein oxidation due to its ability to modulate HOCl synthesis in the MPO active site.
Topics: Albumins; Antioxidants; Ceruloplasmin; Humans; Oxidation-Reduction; Peroxidase; Tyrosine
PubMed: 31238865
DOI: 10.1134/S0006297919060087 -
ACS Chemical Biology May 2009Combinatorial libraries built with severely restricted chemical diversity have yielded highly functional synthetic binding proteins. Structural analyses of these... (Review)
Review
Combinatorial libraries built with severely restricted chemical diversity have yielded highly functional synthetic binding proteins. Structural analyses of these minimalist binding sites have revealed the dominant role of large tyrosine residues for mediating molecular contacts and of small serine/glycine residues for providing space and flexibility. The concept of using limited residue types to construct optimized binding proteins mirrors findings in the field of small molecule drug development, where it has been proposed that most drugs are built from a limited set of side chains presented by diverse frameworks. The physicochemical properties of tyrosine make it the amino acid that is most effective for mediating molecular recognition, and protein engineers have taken advantage of these characteristics to build tyrosine-rich protein binding sites that outperform natural proteins in terms of affinity and specificity. Knowledge from preceding studies can be used to improve current designs, and thus synthetic protein libraries will continue to evolve and improve. In the near future, it seems likely that synthetic binding proteins will supersede natural antibodies for most purposes, and moreover, synthetic proteins will enable many new applications beyond the scope of natural proteins.
Topics: Binding Sites; Crystallography, X-Ray; Drug Design; Models, Molecular; Molecular Structure; Peptide Library; Protein Conformation; Tyrosine
PubMed: 19298050
DOI: 10.1021/cb800314v -
Nature Communications Sep 2021Protein serine/threonine/tyrosine (S/T/Y) phosphorylation is an essential and frequent post-translational modification in eukaryotes, but historically has been...
Protein serine/threonine/tyrosine (S/T/Y) phosphorylation is an essential and frequent post-translational modification in eukaryotes, but historically has been considered less prevalent in bacteria because fewer proteins were found to be phosphorylated and most proteins were modified to a lower degree. Recent proteomics studies greatly expanded the phosphoproteome of Escherichia coli to more than 2000 phosphorylation sites (phosphosites), yet mechanisms of action were proposed for only six phosphosites and fitness effects were described for 38 phosphosites upon perturbation. By systematically characterizing functional relevance of S/T/Y phosphorylation in E. coli metabolism, we found 44 of the 52 mutated phosphosites to be functional based on growth phenotypes and intracellular metabolome profiles. By effectively doubling the number of known functional phosphosites, we provide evidence that protein phosphorylation is a major regulation process in bacterial metabolism. Combining in vitro and in vivo experiments, we demonstrate how single phosphosites modulate enzymatic activity and regulate metabolic fluxes in glycolysis, methylglyoxal bypass, acetate metabolism and the split between pentose phosphate and Entner-Doudoroff pathways through mechanisms that include shielding the substrate binding site, limiting structural dynamics, and disrupting interactions relevant for activity in vivo.
Topics: Binding Sites; Enzymes; Escherichia coli; Escherichia coli Proteins; Mass Spectrometry; Metabolomics; Mutation; Phosphorylation; Protein Processing, Post-Translational; Proteome; Proteomics; Serine; Threonine; Tyrosine
PubMed: 34561442
DOI: 10.1038/s41467-021-25988-4 -
Nature Feb 2022Aromatic residues cluster in the core of folded proteins, where they stabilize the structure through multiple interactions. Nuclear magnetic resonance (NMR) studies in...
Aromatic residues cluster in the core of folded proteins, where they stabilize the structure through multiple interactions. Nuclear magnetic resonance (NMR) studies in the 1970s showed that aromatic side chains can undergo ring flips-that is, 180° rotations-despite their role in maintaining the protein fold. It was suggested that large-scale 'breathing' motions of the surrounding protein environment would be necessary to accommodate these ring flipping events. However, the structural details of these motions have remained unclear. Here we uncover the structural rearrangements that accompany ring flipping of a buried tyrosine residue in an SH3 domain. Using NMR, we show that the tyrosine side chain flips to a low-populated, minor state and, through a proteome-wide sequence analysis, we design mutants that stabilize this state, which allows us to capture its high-resolution structure by X-ray crystallography. A void volume is generated around the tyrosine ring during the structural transition between the major and minor state, and this allows fast flipping to take place. Our results provide structural insights into the protein breathing motions that are associated with ring flipping. More generally, our study has implications for protein design and structure prediction by showing how the local protein environment influences amino acid side chain conformations and vice versa.
Topics: Crystallography, X-Ray; Motion; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Proteins; Tyrosine; src Homology Domains
PubMed: 35173330
DOI: 10.1038/s41586-022-04417-6 -
Biochimica Et Biophysica Acta.... Oct 2022Post-translational modifications and naturally occurring mutations of cytochrome c have been recognized as a regulatory mechanism to control its biology. In this work,...
Post-translational modifications and naturally occurring mutations of cytochrome c have been recognized as a regulatory mechanism to control its biology. In this work, we investigate the effect of such in vivo chemical modifications of human cytochrome c on its redox properties in the adsorbed state onto an electrode. In particular, tyrosines 48 and 97 have been replaced by the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF), thus mimicking tyrosine phosphorylation. Additionally, tyrosine 48 has been replaced by a histidine producing the natural Y48H pathogenic mutant. Thermodynamics and kinetics of the interfacial electron transfer of wild-type cytochrome c and herein produced variants, adsorbed electrostatically under different local interfacial electric fields, were determined by means of variable temperature cyclic film voltammetry. It is shown that non-native cytochrome c variants immobilized under a low interfacial electric field display redox thermodynamics and kinetics similar to those of wild-type cytochrome c. However, upon increasing the strength of the electric field, the redox thermodynamics and kinetics of the modified proteins markedly differ from those of the wild-type species. The mutations promote stabilization of the oxidized form and a significant increase in the activation enthalpy values that can be ascribed to a subtle distortion of the heme cofactor and/or difference of the amino acid rearrangements rather than to a coarse protein structural change. Overall, these results point to a combined effect of the single point mutations at positions 48 and 97 and the strength of electrostatic binding on the regulatory mechanism of mitochondrial membrane activity, when acting as a redox shuttle protein.
Topics: Cytochromes c; Electrodes; Humans; Oxidation-Reduction; Thermodynamics; Tyrosine
PubMed: 35643148
DOI: 10.1016/j.bbabio.2022.148570 -
Biomacromolecules Mar 2022The modulation of reaction kinetics with horseradish peroxidase (HRP)-catalyzed cross-linking of proteins remains a useful strategy to modulate hydrogel formation. Here,...
The modulation of reaction kinetics with horseradish peroxidase (HRP)-catalyzed cross-linking of proteins remains a useful strategy to modulate hydrogel formation. Here, we demonstrate that the presence of positively charged lysines in silk-elastin-like polymers impacts the thermal transition temperature of these proteins, while the location in the primary sequence modulates the reactivity of the tyrosines. The positively charged lysine side chains decreased π-π interactions among the tyrosines and reduced the rate of formation and number of HRP-mediated dityrosine bonds, dependent on the proximity of the charged group to the tyrosine. The results suggest that the location of repulsive charges can be used to tailor the reaction kinetics for enzymatic cross-linking, providing further control of gelation rates for gel formation and the resulting protein-based gel characteristics.
Topics: Cross-Linking Reagents; Elastin; Horseradish Peroxidase; Hydrogels; Silk; Tyrosine
PubMed: 35113522
DOI: 10.1021/acs.biomac.1c01192