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Title: Peptide ligands specific to the oxidized form of escherichia coli thioredoxin.

Abstract

Thioredoxin (Trx) is a highly conserved redox protein involved in several essential cellular processes. In this study, our goal was to isolate peptide ligands to Escherichia coli Trx that mimic protein-protein interactions, specifically the T7 polymerase-Trx interaction. To do this, we subjected Trx to affinity selection against a panel of linear and cysteine-constrained peptides using M13 phage display. A novel cyclized conserved peptide sequence, with a motif of C(D/N/S/T/G)D(S/T)-hydrophobic-C-X-hydrophobic-P, was isolated to Trx. These peptides bound specifically to the E. coli Trx when compared to the human and spirulina homologs. An alanine substitution of the active site cysteines (CGPC) resulted in a significant loss of peptide binding affinity to the Cys-32 mutant. The peptides were also characterized in the context of Trx's role as a processivity factor of the T7 DNA polymerase (gp5). As the interaction between gp5 and Trx normally takes place under reducing conditions, which might interfere with the conformation of the disulfide-bridged peptides, we made use of a 22 residue deletion mutant of gp5 in the thioredoxin binding domain (gp5{Delta}22) that bypassed the requirements of reducing conditions to interact with Trx. A competition study revealed that the peptide selectively inhibits the interaction of gp5{Delta}22 with Trx, undermore » oxidizing conditions, with an IC50 of {approx} 10 {micro}M.« less

Authors:
; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
992390
Report Number(s):
ANL/BIO/JA-62337
Journal ID: ISSN 0006-3002; BBACAQ; TRN: US201022%%291
DOE Contract Number:
DE-AC02-06CH11357
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochim. et Biophysica Acta; Journal Volume: 1784; Journal Issue: Nov. 2008
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; AFFINITY; ALANINES; DNA POLYMERASES; ESCHERICHIA COLI; MUTANTS; PEPTIDES; PROTEINS; RESIDUES

Citation Formats

Scholle, M. D., Banach, B. S., Hamdan, S. M., Richardson, C. C., Kay, B. K., Biosciences Division, Amunix, Inc., Univ. of Illinois at Chicago, and Harvard Medical School. Peptide ligands specific to the oxidized form of escherichia coli thioredoxin.. United States: N. p., 2008. Web. doi:10.1016/j.bbapap.2008.06.022.
Scholle, M. D., Banach, B. S., Hamdan, S. M., Richardson, C. C., Kay, B. K., Biosciences Division, Amunix, Inc., Univ. of Illinois at Chicago, & Harvard Medical School. Peptide ligands specific to the oxidized form of escherichia coli thioredoxin.. United States. doi:10.1016/j.bbapap.2008.06.022.
Scholle, M. D., Banach, B. S., Hamdan, S. M., Richardson, C. C., Kay, B. K., Biosciences Division, Amunix, Inc., Univ. of Illinois at Chicago, and Harvard Medical School. Sat . "Peptide ligands specific to the oxidized form of escherichia coli thioredoxin.". United States. doi:10.1016/j.bbapap.2008.06.022.
@article{osti_992390,
title = {Peptide ligands specific to the oxidized form of escherichia coli thioredoxin.},
author = {Scholle, M. D. and Banach, B. S. and Hamdan, S. M. and Richardson, C. C. and Kay, B. K. and Biosciences Division and Amunix, Inc. and Univ. of Illinois at Chicago and Harvard Medical School},
abstractNote = {Thioredoxin (Trx) is a highly conserved redox protein involved in several essential cellular processes. In this study, our goal was to isolate peptide ligands to Escherichia coli Trx that mimic protein-protein interactions, specifically the T7 polymerase-Trx interaction. To do this, we subjected Trx to affinity selection against a panel of linear and cysteine-constrained peptides using M13 phage display. A novel cyclized conserved peptide sequence, with a motif of C(D/N/S/T/G)D(S/T)-hydrophobic-C-X-hydrophobic-P, was isolated to Trx. These peptides bound specifically to the E. coli Trx when compared to the human and spirulina homologs. An alanine substitution of the active site cysteines (CGPC) resulted in a significant loss of peptide binding affinity to the Cys-32 mutant. The peptides were also characterized in the context of Trx's role as a processivity factor of the T7 DNA polymerase (gp5). As the interaction between gp5 and Trx normally takes place under reducing conditions, which might interfere with the conformation of the disulfide-bridged peptides, we made use of a 22 residue deletion mutant of gp5 in the thioredoxin binding domain (gp5{Delta}22) that bypassed the requirements of reducing conditions to interact with Trx. A competition study revealed that the peptide selectively inhibits the interaction of gp5{Delta}22 with Trx, under oxidizing conditions, with an IC50 of {approx} 10 {micro}M.},
doi = {10.1016/j.bbapap.2008.06.022},
journal = {Biochim. et Biophysica Acta},
number = Nov. 2008,
volume = 1784,
place = {United States},
year = {Sat Nov 01 00:00:00 EDT 2008},
month = {Sat Nov 01 00:00:00 EDT 2008}
}
  • Escherichia coli thioredoxin (M/sub r/ 11,700) usually functions as a hydrogen carrier protein that undergoes reversible oxidation/reduction reactions of its active-site disulfide linkage. By use of a number of assigned and identified resonances in one- and two-dimensional /sup 1/H NMR spectra, the two forms of the protein have been compared. Only groups that are relatively close to the active-site Cys-32, Cys-35 linkage such as Trp-28, Trp-31, Phe-27, Ala-29, and Val-25 undergo substantial changes in their /sup 1/H NMR chemical shift upon reduction. Thus, the structural changes that occur upon reduction appear to be localized to the disulfide-containing turn and themore » central strand of the twisted ..beta..-sheet that directly leads to this turn. Notwithstanding the apparent similarity in the secondary and tertiary structures of the oxidized and reduced forms of the protein, the thermal stability of the protein decreases by 10/sup 0/C upon the reduction of the single disulfide. This was found by both /sup 1/H NMR and near- and far-ultraviolet circular dichroism studies. Oxidized thioredoxin was also more resistant to alkaline denaturation. Furthermore, the exchange rate of the relatively stable slow-exchanging backbone amide protons that are part of the core of the twisted five-stranded ..beta..-sheet of thioredoxin increases substantially after reduction. These findings are discussed with reference to the reported effects of disulfides on the thermal stability of proteins.« less
  • The three-dimensional solution structure of reduced (dithiol) thioredoxin from Escherichia coli has been determined with distance and dihedral angle constraints obtained from {sup 1}H NMR spectroscopy. Reduced thioredoxin has a well-defined global fold consisting of a central five-strand {beta}-sheet and three long helices. The {beta}-strands are packed in the sheet in the order {beta}{sub 1}{beta}{sub 3}{beta}{sub 2}{beta}{sub 4}{beta}{sub 5}, with {beta}{sub 1}, {beta}{sub 3}, and {beta}{sub 2} parallel and {beta}{sub 2}, {beta}{sub 4}, and {beta}{sub 5} arranged in an antiparallel fashion. Two of the helices connect strands of the {beta}-sheet: {alpha}{sub 1} between {beta}{sub 1} and {beta}{sub 2} and {alpha}{submore » 2} between {beta}{sub 2} and {beta}{sub 3}. Strands {beta}{sub 4} and {beta}{sub 5} are connected by a short loop that contains a {beta}-bulge. Strands {beta}{sub 3} and {beta}{sub 4} are connected by a long loop that contains a series of turn-like or 3{sub 10} helical structures. The active site Cys-Gly-Pro-Cys sequence forms a protruding loop between strand {beta}{sub 2} and helix {alpha}{sub 2}. The structure is very similar overall to that of oxidized (disulfide) thioredoxin obtained from X-ray crystal structure analysis but differs in the local conformation of the active site loop. The distance between the sulfurs of Cys 32 and Cys 35 increases from 2.05 {angstrom} in the disulfide bridge to 6.8 {plus minus} 0.6 {angstrom} in the dithiol of reduced thioredoxin, as a result of a rotation of the side chain of Cys 35 and a significant change in the position of Pro 34. This conformational change has important implications for the mechanism of thioredoxin as a protein disulfide oxidoreductase.« less
  • The structure of the 142-residue protein Q8ZP25 SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE ECOLI encoded in the genome of Escherichia coli was determined by NMR. The two proteins belong to Pfam (Finn et al. 34:D247-D251, 2006) PF07449, which currently comprises 50 members, and belongs itself to the 'thioredoxin-like clan'. However, protein HYAE ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE ECOLI was previously classified as a (NiFe)more » hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides.« less
  • In order to obtain more insight in the initial steps of the process of protein translocation across membranes, biophysical investigations were undertaken on the lipid specificity and structural consequences of penetration of the PhoE signal peptide into lipid model membranes and on the conformation of the signal peptide adopted upon interaction with the lipids. When the monolayer technique and differential scanning calorimetry are used, a stronger penetration is observed for negatively charged lipids, significantly influenced by the physical state of the lipid but not by temperature or acyl chain unsaturation as such. Although the interaction is principally electrostatic, as indicatedmore » also by the strong penetration of N-terminal fragments into negatively charged lipid monolayers, the effect of ionic strength suggests an additional hydrophobic component. Most interestingly with regard to the mechanism of protein translocation, the molecular area of the peptide in the monolayer also shows lipid specificity: the area in the presence of PC is consistent with a looped helical orientation, whereas in the presence of cardiolipin a time-dependent conformational change is observed, most likely leading from a looped to a stretched orientation with the N-terminus directed toward the water. This is in line also with the determined peptide-lipid stoichiometry. Preliminary /sup 31/P NMR and electron microscopy data on the interaction with lipid bilayer systems indicate loss of bilayer structure.« less
  • Complete proton assignments are reported for the {sup 1}H nuclear magnetic resonance (NMR) spectrum of Escherichia coli thioredoxin in the oxidized (with active-site disulfide bridge) and reduced (with two sulfhydryl groups) states. The assignments were obtained by using an integrated assignment strategy in which spin systems were identified from a combination of relayed and multiple quantum NMR techniques prior to sequential assignment. Elements of secondary structure were identified in each protein from characteristic nuclear Overhauser effects (NOE), coupling constants, and slowly exchanging amide protons. In both oxidized and reduced thioredoxin, approximately 33% of the 108 amino acid residues participate inmore » a {beta}-sheet containing four major strands (three antiparallel and one parallel). The global folds of oxidized and reduced thioredoxin are shown to be essentially identical. Both NOE connectivities and chemical shift values for the two proteins are very similar, except in the immediate vicinity of the active site where significant variations in the chemical shift indicate subtle conformational changes. While the overall fold of oxidized thioredoxin is the same in solution and in the crystalline state, some small differences in local conformation are apparent.« less