Application of High-Throughput Competition Experiments in the Development of Aspartate-Directed Site-Selective Modification of Tyrosine Residues in Peptides.
Ontology highlight
ABSTRACT:
SUBMITTER: Chinn AJ
PROVIDER: S-EPMC7966973 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:We report here a photochemical process for the selective modification of tryptophan (Trp) residues in peptides and small proteins using electron-responsive N-carbamoylpyridinium salts and UV-B light. Preliminary mechanistic experiments suggest that the photoconjugation process proceeds through photoinduced electron transfer (PET) between Trp and the pyridinium salt, followed by fragmentation of the pyridinium N-N bond and concomitant transfer of this group to Trp. The reaction displays excellent site selectivity for Trp and is tolerant to other, redox-active amino-acid residues. Moreover, the reaction proceeds in pure aqueous conditions without the requirement of organic cosolvents or photocatalysts, is enhanced by glutathione, and operates efficiently over a wide range of peptide concentrations (10-700 ?M). The scope of the process was explored through the labeling of 6-Trp-containing peptides and proteins ranging from 1 to 14 kDa. We demonstrate the versatility of the N-carbamoylpyridinium salt both by tuning the electrochemical and photochemical properties of the pyridinium scaffold to enable challenging photoconjugation reactions and by using the carbamoyl moiety to tether a plethora of productive functional groups, including reactive handles, purification tags, and removable protecting groups.
| S-EPMC7292481 | biostudies-literature
Project description:We report the late-stage chemical modification of ribosomally synthesized and post-translationally modified peptides (RIPPs) by Diels-Alder cycloadditions to naturally occurring dehydroalanines. The tail region of the thiopeptide thiostrepton could be modified selectively and efficiently under microwave heating and transition-metal-free conditions. The Diels-Alder adducts were isolated and the different site- and endo/exo isomers were identified by 1D/2D 1 H NMR. Via efficient modification of the thiopeptide nosiheptide and the lanthipeptide nisin Z the generality of the method was established. Minimum inhibitory concentration (MIC) assays of the purified thiostrepton Diels-Alder products against thiostrepton-susceptible strains displayed high activities comparable to that of native thiostrepton. These Diels-Alder products were also subjected successfully to inverse-electron-demand Diels-Alder reactions with a variety of functionalized tetrazines, demonstrating the utility of this method for labeling of RiPPs.
| S-EPMC6790694 | biostudies-literature
Project description:Prediction methods as well as experimental methods for T-cell epitope discovery have developed significantly in recent years. High-throughput experimental methods have made it possible to perform full-length protein scans for epitopes restricted to a limited number of MHC alleles. The high costs and limitations regarding the number of proteins and MHC alleles that are feasibly handled by such experimental methods have made in silico prediction models of high interest. MHC binding prediction methods are today of a very high quality and can predict MHC binding peptides with high accuracy. This is possible for a large range of MHC alleles and relevant length of binding peptides. The predictions can easily be performed for complete proteomes of any size. Prediction methods are still, however, dependent on good experimental methods for validation, and should merely be used as a guide for rational epitope discovery. We expect prediction methods as well as experimental validation methods to continue to develop and that we will soon see clinical trials of products whose development has been guided by prediction methods.
| S-EPMC3297080 | biostudies-literature
Project description:To elucidate the reaction mechanism of xylanase, the identification of amino acids essential for its catalysis is of importance. Studies have indicated the possibility that the reaction mechanism of xylanase is similar to that of hen's egg lysozyme, which involves acidic amino acid residues. On the basis of this assumption, together with the three-dimensional structure of Bacillus pumilus xylanase and its amino acid sequence similarity to other xylanases of different origins, three acidic amino acids, namely Asp-21, Glu-93 and Glu-182, were selected for site-directed mutagenesis. The Asp residue was altered to either Ser or Glu, and the Glu residues to Ser or Asp. The purified mutant xylanases D21E, D21S, E93D, E93S, E182D and E182S showed single protein bands of about 26 kDa on SDS/PAGE. C.d. spectra of these mutant enzymes show no effect on the secondary structure of xylanase, except that of D21E, which shows a little variation. Furthermore, mutations of Glu-93 and Glu-182 resulted in a drastic decrease in the specific activity of xylanase as compared with mutation of Asp-21. On the basis of these results we propose that Glu-93 and Glu-182 are the best candidates for the essential catalytic residues of xylanase.
| S-EPMC1132087 | biostudies-other
Project description:Site-selective protein-protein coupling has long been a goal of chemical biology research. In recent years, that goal has been realized to varying degrees through a number of techniques, including the use of tyrosinase-based coupling strategies. Early publications utilizing tyrosinase from Agaricus bisporus(abTYR) showed the potential to convert tyrosine residues into ortho-quinone functional groups, but this enzyme is challenging to produce recombinantly and suffers from some limitations in substrate scope. Initial screens of several tyrosinase candidates revealed that the tyrosinase from Bacillus megaterium (megaTYR) is an enzyme that possesses a broad substrate tolerance. We use the expanded substrate preference as a starting point for protein design experiments and show that single point mutants of megaTYR are capable of activating tyrosine residues in various sequence contexts. We leverage this new tool to enable the construction of protein trimers via a charge-directed sequential activation of tyrosine residues (CDSAT).
| S-EPMC9274618 | biostudies-literature
Project description:Myostatin, a negative regulator of skeletal muscle growth, is a promising target for treating muscle atrophic disorders. Recently, we discovered a minimal myostatin inhibitor 1 (WRQNTRYSRIEAIKIQILSKLRL-amide) derived from positions 21-43 of the mouse myostatin prodomain. We previously identified key residues (N-terminal Trp21, rodent-specific Tyr27, and all aliphatic amino acids) required for effective inhibition through structure-activity relationship (SAR) studies based on 1 and characterized a 3-fold more potent inhibitor 2 bearing a 2-naphthyloxyacetyl group at position 21. Herein, we performed 1-based SAR studies focused on all aliphatic residues and Ala32, discovering that the incorporations of Trp and Ile at positions 32 and 38, respectively, enhanced the inhibitory activity. Combining these findings with 2, a novel peptide 3d displayed an IC50 value of 0.32 ?M, which is 11 times more potent than 1. The peptide 3d would have the potential to be a promising drug lead to develop better peptidomimetics.
| S-EPMC5512132 | biostudies-literature
Project description:Treatment of the purple membrane of Halobacterium halobium with tetranitromethane led to modification of tyrosine residues. Modification of more than 3-4 tyrosine residues per bacteriorhodopsin monomer caused a decrease in the light-induced proton-pumping ability of purple membrane in synthetic lipid vesicles, loss of the sharp X-ray-diffraction patterns characteristic of the crystal lattice, loss of the absorbance maximum at 560 nm, and change in the buoyant density of the membrane. No modification of lipid was detected. These changes were interpreted as a gradual denaturation of the protein component such that when 8-9 tyrosine residues are modified, no proton pumping is observed. Modification of less than 3-4 tyrosine residues with tetranitromethane caused an increse in light-induced proton pumping. It was possible to generate partly modified purple membrane which had completely lost the property of diffracting X-rays into the sharp pattern observed with native purple membrane, but which still retained the ability to pump protons in a vectorial manner. Retention of crystal lattice is not essential for proton pumping.
| S-EPMC1186614 | biostudies-other
Project description:Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear Tb(III) complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr), have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the Tb(III) ion as the emission center. Even in the coexistence of phosphoserine (pSer) and phosphothreonine (pThr), pTyr can be efficintly detected with high selectivity. Simply by adding these Tb(III) complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates.
| S-EPMC4916314 | biostudies-literature
Project description:Selective derivatization of solvent-exposed cysteine residues in peptides and proteins is achieved by brief irradiation of an aqueous solution containing 3-(hydroxymethyl)-2-naphthol derivatives (NQMPs) with 350 nm fluorescent lamp. NQMP can be conjugated with various moieties, such as PEG, dyes, carbohydrates, or possess a fragment for further selective derivatization, e.g., biotin, azide, alkyne, etc. Attractive features of this labeling approach include an exceptionally fast rate of the reaction and a requirement for low equivalence of the reagent. The NQMP-thioether linkage is stable under ambient conditions, survives protein digestion and MS analysis. Irradiation of NQMP-labeled protein in a dilute solution (<40 ?M) or in the presence of a vinyl ether results in a traceless release of the substrate. The reversible biotinylation of bovine serum albumin, as well as capture and release of this protein using NeutrAvidin Agarose resin beads has been demonstrated.
| S-EPMC3994131 | biostudies-literature
Project description:We previously reported that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used to selectively derivatize thiols for mass spectrometric analysis, and the introduced selenium tags are useful as they could survive or removed with collision-induced dissociation (CID). Described herein is the further study of the reactivity of various protein/peptide thiols toward NPSP and its application to derivatize thiol peptides in protein digests. With a modified protocol (i.e., dissolving NPSP in acetonitrile instead of aqueous solvent), we found that quantitative conversion of thiols can be obtained in seconds, using NPSP in a slight excess amount (NPSP:thiol of 1.1-2:1). Further investigation shows that the thiol reactivity toward NPSP reflects its chemical environment and accessibility in proteins/peptides. For instance, adjacent basic amino acid residues increase the thiol reactivity, probably because they could stabilize the thiolate form to facilitate the nucleophilic attack of thiol on NPSP. In the case of creatine phosphokinase, the native protein predominately has one thiol reacted with NPSP while all of four thiol groups of the denatured protein can be derivatized, in accordance with the corresponding protein conformation. In addition, thiol peptides in protein/peptide enzymatic digests can be quickly and effectively tagged by NPSP following tri-n-butylphosphine (TBP) reduction. Notably, all three thiols of the peptide QCCASVCSL in the insulin peptic digest can be modified simultaneously by NPSP. These results suggest a novel and selective method for protecting thiols in the bottom-up approach for protein structure analysis.
| S-EPMC3704154 | biostudies-literature