Browse
Submit Data
Databases
API
Help

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Application of High-Throughput Competition Experiments in the Development of Aspartate-Directed Site-Selective Modification of Tyrosine Residues in Peptides.

ABSTRACT: Herein we report a Cu-catalyzed, site-selective functionalization of peptides that employs an aspartic acid (Asp) as a native directing motif, which directs the site of O-arylation at a proximal tyrosine (Tyr) residue. Through a series of competition studies conducted in high-throughput reaction arrays, effective conditions were identified that gave high selectivity for the proximal Tyr in Asp-directed Tyr modification. Good levels of site-selectivity were achieved in the O-arylation at a proximal Tyr residue in a number of cases, including a peptide-small molecule hybrid.

SUBMITTER: Chinn AJ

PROVIDER: S-EPMC7966973 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus.

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

Aspartate Residues Far From the Active Site Drive O-GlcNAc Transferase Substrate Selection

Project description:Abstract: O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions.

2019-07-24 | GSE132205 | GEO

Role of cysteine residues in ribonuclease H from Escherichia coli. Site-directed mutagenesis and chemical modification.

Project description:The role of the three cysteine residues at positions 13, 63 and 133 in Escherichia coli RNAase H, an enzyme that is sensitive to N-ethylmaleimide [Berkower, Leis & Hurwitz (1973) J. Biol. Chem. 248, 5914-5921], was examined by using both site-directed mutagenesis and chemical modification. Novel aspects that were found are as follows. First, none of the cysteine residues is required for activity. Secondly, chemical modification of either Cys-13 or Cys-133 with thiol-blocking reagents inactivates the enzyme, but that of Cys-63 does not. Thus the sensitivity of E. coli RNAase H to N-ethylmaleimide arises not from blocking of the thiol group but from steric hindrance by the modifying group incorporated at either Cys-13 or Cys-133.

| S-EPMC1150208 | biostudies-other

Site-directed mutagenesis and chemical modification of cysteine residues of rat glutathione S-transferase 3-3.

Project description:Rat liver glutathione S-transferase (GST) 3-3 is composed of two identical subunits, each containing three cysteine residues, Cys-86, Cys-114 and Cys-173. We have shown previously that Cys-86 is not involved in the enzymic activity of GST 3-3 [Hsieh, Huang, Chen, Lai & Tam (1991) Biochem, J. 278, 293-297]. At 50 degrees C, iodoacetamide can inactivate the enzyme by modifying Cys-86 and Cys-114. Cys-114 can be protected against iodoacetamide inhibition by S-(dinitrophenyl)glutathione. Site-directed mutagenesis was used to construct mutants in which serine replaced one (C114S and C173S) or all three (CallS) cysteine residues. These mutants were over-expressed in Spodoptera frugiperda cells in a baculovirus system and were found to be fully active. Replacing Cys-86 or Cys-114 with alanine (C86A and C114A) does not diminish the activity of the protein. The results suggest that cysteines are not involved in the enzymic mechanism, and Cys-114 is possibly located at the active site of GST 3-3.

| S-EPMC1133040 | biostudies-other

Predictions versus high-throughput experiments in T-cell epitope discovery: competition or synergy?

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

Aspartate Residues Far from the Active Site Drive O-GlcNAc Transferase Substrate Selection.

Project description:O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions.

| S-EPMC6849375 | biostudies-literature

Synthesis of Multi-Protein Complexes through Charge-Directed Sequential Activation of Tyrosine Residues.

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

Development of Potent Myostatin Inhibitory Peptides through Hydrophobic Residue-Directed Structural Modification.

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

Aspartate residues far from the active site drive O-GlcNAc transferase substrate selection

Project description:Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues Cterminal to the glycosite. In addition to guiding design of inhibitors that target OGT’s TPR domain, this information will inform efforts to engineer substrates to explore biological functions.

2019-07-26 | PXD014731 | Pride

Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin.

Project description:Redox active cysteine residues including ?Cys93 are part of hemoglobin's "oxidation hotspot". Irreversible oxidation of ?Cys93 ultimately leads to the collapse of the hemoglobin structure and release of heme. Human fetal hemoglobin (HbF), similarly to the adult hemoglobin (HbA), carries redox active ?Cys93 in the vicinity of the heme pocket. Site-directed mutagenesis has been used in this study to examine the impact of removal and/or addition of cysteine residues in HbF. The redox activities of the recombinant mutants were examined by determining the spontaneous autoxidation rate, the hydrogen peroxide induced ferric to ferryl oxidation rate, and irreversible oxidation of cysteine by quantitative mass spectrometry. We found that substitution of ?Cys93Ala resulted in oxidative instability characterized by increased oxidation rates. Moreover, the addition of a cysteine residue at ?19 on the exposed surface of the ?-chain altered the regular electron transfer pathway within the protein by forming an alternative oxidative site. This may also create an accessible site for di-sulfide bonding between Hb subunits. Engineering of cysteine residues at suitable locations may be useful as a tool for managing oxidation in a protein, and for Hb, a way to stave off oxidation reactions resulting in a protein structural collapse.

| S-EPMC6126208 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data