scholarly journals Converting disulfide bridges in native peptides to stable methylene thioacetals

2016 ◽  
Vol 7 (12) ◽  
pp. 7007-7012 ◽  
Author(s):  
C. M. B. K. Kourra ◽  
N. Cramer
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bridges ◽  
Simple Protocol ◽  
Native Peptides

A mild and simple protocol converts the labile disulfide bond of unprotected native peptides into highly stable methylene thioacetals, annihilating reductive lability and increasing stability.

Determination with Matrix-Assisted Laser Desorption/Ionization Tandem Time-of-Flight Mass Spectrometry of the Extensive Disulfide Bonding in Tarantula Venom Peptide Psalmopeotoxin I

2009 ◽  
Vol 15 (4) ◽  
pp. 517-529 ◽  
Author(s):  
Audrey Combes ◽  
Soo Jin Choi ◽  
Cyril Pimentel ◽  
Hervé Darbon ◽  
Dietmar Waidelich ◽  
...  
Keyword(s):  
Laser Desorption ◽  
Disulfide Bridge ◽  
Fragmentation Pattern ◽  
Laser Desorption Ionization ◽  
Disulfide Bridges ◽  
Wild Type ◽  
Desorption Ionization ◽  
Native Peptides ◽  
Matrix Assisted Laser ◽  
Matrix Assisted Laser Desorption

Psalmopeotoxin I (PcFK1) is a 33-residue peptide isolated from the venom of the tarantula Psalmopoeus cambridgei. This peptide specifically inhibits the intra-erythrocyte stage of Plasmodium falciparum in vitro. It contains six cysteine residues forming three disulfide bridges and belongs to the superfamily of natural peptides containing the inhibitor cystine knot (ICK) fold. We produced the wild-type and mutated forms of the recombinant peptide to examine the mechanism of action of PcFK1. The purified toxins were consistently produced as two isobaric peptides (r-PcFK1-1 and r-PcFK1-2) with different retention properties but identical anti-plasmodial biological activity. Comparison of 15N-NMR heteronuclear single quantum correlation spectra revealed that although rPcFK1-1 was highly structured, rPcFK1-2 does not have a stable three-dimensional structure. We used high-energy collision-induced fragmentation of the peptides with a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer to further investigate the structure of the native peptides in its natural form and produced in E. coli. The fragmentation spectra of the native peptides were very complex due to the occurrence in the spectrum of ions resulting from (1) cross-linking of fragments through a disulfide bridge and (2) asymmetric fragmentations of the disulfide bridges and (3) multiple neutral losses. The tandem mass spectrometry fragmentation pattern of r-PcFK1-1 was similar to that of the natural peptide isolated from crude venom, but r-PcFK1-2 had a clearly distinct fragmentation pattern, more closely resembling the fragmentation spectra of reduced and alkylated peptides. Observed ions could be attributed to specific fragments by comparing spectra between the wild-type and selected variants with point mutations (Y11W, R20T, Y26W, K28V). The disulfide connections in r-PcFK1-2 differed from those of the native peptide and showed a rare disulfide bridge between vicinal cysteine residues. The r-PcFK1_(R20T) variant showed a very limited fragmentation pattern when analyzed in positive mode but displayed much more fragmentation in negative mode pointing out the importance of the R20 residue in the fragmentation of PcFK1. Using the reductive matrix 1,5-diaminonaphtalene promoted strongly in source decay fragmentation of the peptides in MS mode. Our findings illustrated the critical role of the electronic environment around the central Cys18–Cys19 doublet in PcFK1 in internal fragmentation of the peptide.

Fabrication of Magnetic Upconversion Nanohybrid for Luminescent Resonance Energy Transfer-Based Detection of Glutathione

2015 ◽  
Vol 15 (10) ◽  
pp. 7950-7954 ◽  
Author(s):  
Phuong-Diem Nguyen ◽  
Vu Thanh Cong ◽  
Sang Jun Son ◽  
Junhong Min
Keyword(s):  
Energy Transfer ◽  
Magnetic Separation ◽  
Disulfide Bond ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Silica Matrix ◽  
Upconversion Nanoparticles ◽  
Disulfide Bridges ◽  
Detection Mechanism ◽  
Facile Fabrication

This study introduces the facile fabrication of a bimodal nanohybrid for the luminescent ON/OFF detection of glutathione. The proposed nanosensor consists of magnetic (Fe3O4) and upconversion nanoparticles (UCP) co-encapsulated in a silica matrix, and decorated with gold nanoparticle (AuNP) as a luminescent quencher. The detection mechanism is based on the Luminescent Resonance Energy Transfer (LRET) between the donor (UCP) and the acceptor (AuNP) with the help of a disulfide bond as a bridging element. In the presence of glutathione, the disulfide bridges between AuNPs and Fe3O4/UCP@SiO2 was cleaved and the amount of glutathione could be traced by the restored luminescence (ON state) of the nanohybrids after magnetic separation.

scholarly journals Disulfide Bridges Are Not Involved in Penicillin-Binding Protein 1b Dimerization in Escherichia coli

1999 ◽  
Vol 181 (9) ◽  
pp. 2970-2972 ◽  
Author(s):  
Christian Chalut ◽  
Marie-Hélène Remy ◽  
Jean-Michel Masson
Keyword(s):  
Escherichia Coli ◽  
Disulfide Bond ◽  
Binding Protein ◽  
Deletion Mutants ◽  
Disulfide Bridges ◽  
Penicillin Binding Protein ◽  
Intermolecular Disulfide Bond

ABSTRACT PBP1b can be found as a dimer in Escherichia coli. Previous results suggested that dimerization involved the cysteine(s) in an intermolecular disulfide bond. We show that either deletion mutants or a mutant without cysteines is fully active and still binds penicillin and that the latter can also form dimers.

The Disulfide Bond In Antithrombin Required For Interaction With Heparin

1981 ◽  
Author(s):  
T H Finlay ◽  
W S Ferguson
Keyword(s):  
Disulfide Bond ◽  
Preliminary Analysis ◽  
Fluorescence Enhancement ◽  
Sequence Data ◽  
Tryptophan Fluorescence ◽  
Order Rate Constant ◽  
Disulfide Bridges ◽  
Research Career ◽  
Intrinsic Tryptophan Fluorescence ◽  
Mild Reduction

Mild reduction of antithrombin (AT) with dithiothreitol followed by alkylation with [14C]-iodoacetamide results in the incorporation of 1.7 mol of acetamide/mol of protein with cysteine the only amino acid modified. Tryptic digestion of the reduced and alkylated AT results in the formation of only two labeled peptides indicating that mild reduction cleaves only one of the 3 disulfide bridges.The 2nd order rate constant for the reaction of thrombin with both native and mildly reduced AT is 5.9-9.6 × 105 M-1 min-1. Addition of heparin accelerates the reaction between native AT and thrombin (k >107 M-1 min-1) but has no effect on the reaction between reduced AT and thrombin (k = 8.3-12.2 ×105 M-1 min-1). Alkylation of reduced AT destroys all remaining thrombin-neutralizing activity.Native AT elutes from a heparin-Sepharose column with 1.0 M NaCl whereas reduced AT elutes at 0.5 M NaCl. Reduced and alkylated AT is not retained by the immobilized heparin even in 0.1 M NaCl. The intrinsic tryptophan fluorescence enhancement observed following binding of heparin to native AT is not seen with reduced AT. These data indicate that reduction of a single disulfide bond in AT lowers the affinity of the protein for heparin and eliminates the heparin-mediated acceleration of thrombin neutralization.Mildly reduced and alkylated AT was digested with CNBr and the peptides were fractionated by HPLC. Preliminary analysis of the CNBr peptides suggests that the crucial disulfide bond is between CYS239 and CYS422 (using the numbering in Magnusson’s sequence data).Supported by grants from the USPHS. THF is a Research Career Development Awardee of the NIH (HL-00277).

Adsorption of Fibrinogen and Fragment D of Fibrinogen onto the Insolubilized αChain of Fibrin

1979 ◽  
Vol 41 (04) ◽  
pp. 687-690
Author(s):  
F R Matthias
Keyword(s):  
Disulfide Bridges ◽  
Soluble Fibrin ◽  
Immobilized Proteins ◽  
Covalently Bound

SummaryAfter thrombin treatment insolubilized fibrinmonomer, which is obtained from insolubilized fibrinogen covalently bound to agarose, adsorbs soluble fibrin and its derivatives from solutions. The immobilized proteins are attached to the agarose by the ‘A’ αchain. After reduction of the disulfide bridges the β and γchains can be removed from the agarose.After thrombin treatment the immobilized αchain adsorbs fibrinogen and fragment D. To some extent the β and γchain do not seem necessary for the adsorption. The amount adsorbed increases, when thrombin treatment of the insolubilized protein follows the reduction process.This may indicate that the fibrinopeptides ‘A’ of the insolubilized αchain are better accessible after the removal of the β and γchains.

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