Rapid Access to ω-Conotoxin Chimeras using Native Chemical Ligation

2009 ◽  
Vol 62 (10) ◽  
pp. 1333 ◽  
Author(s):  
Gene Hopping ◽  
Richard J. Lewis ◽  
Paul F. Alewood
Keyword(s):  
Ligand Binding ◽  
Coupling Efficiency ◽  
Native Chemical Ligation ◽  
Binding Assays ◽  
Chemical Ligation ◽  
Receptor Recognition ◽  
Single Protein ◽  
Difficult Sequences ◽  
Peptide Segments ◽  
Ligation Method

Grafting different regions of related peptides together to form a single protein chimera is a valuable tool in rapidly elucidating regions of activity or selectivity in peptides and proteins. To conveniently evaluate the contributions of the N- and C-terminal segments of ω-conotoxins CVID and MVIIC to activity, we employed native chemical ligation in CVID-MVIIC chimera design. Assembly of these peptide segments via the ligation method improved overall yield and coupling efficiency, with no difficult sequences encountered in contrast to the traditional full-length chain assembly of CVID. Radio-ligand binding assays revealed regions of importance for receptor recognition.

scholarly journals A Straightforward Methodology to Overcome Solubility Challenges for N-Terminal Cysteinyl Peptide Segments Used in Native Chemical Ligation

2020 ◽  
Author(s):  
Skander Abboud ◽  
El hadji Cisse ◽  
Michel Doudeau ◽  
Hélène Bénédetti ◽  
Vincent AUCAGNE
Keyword(s):  
Amino Acids ◽  
Chemical Synthesis ◽  
Building Blocks ◽  
Native Chemical Ligation ◽  
Synthetic Approach ◽  
Chemical Ligation ◽  
Limited Solubility ◽  
Peptide Segments

One of the main limitations encountered during the chemical synthesis of proteins through native chemical ligation (NCL) is the limited solubility of some of the peptide segments. The most commonly used solution to overcome this problem is to derivatize the segment with a temporary solubilizing tag. Conveniently, the tag can be introduced on the thioester segment in such a way that it is removed concomitantly with the NCL reaction. We herein describe a generalization of this approach to N-terminal cysteinyl segment counterparts, using a straightforward synthetic approach that can be easily automated from commercially available building blocks, and applied it to a well-known problematic target, SUMO-2 (93 amino acids).

scholarly journals SEA Ligation Is Accelerated at Mildly Acidic pH. Application to the Formation of Difficult Peptide Junctions

2019 ◽  
Author(s):  
Marine Cargoet ◽  
Vincent Diemer ◽  
Laurent Raibaut ◽  
Elizabeth Lissy ◽  
Benoît Snella ◽  
...  
Keyword(s):  
Total Synthesis ◽  
Peptide Bond ◽  
Native Chemical Ligation ◽  
Acidic Ph ◽  
Chemical Ligation ◽  
Bond Forming ◽  
Neutral Ph ◽  
Wide Range ◽  
Peptide Segments ◽  
Model Peptides

The bis(2-sulfanylethyl)amido (SEA)-mediated ligation has been introduced in 2010 as a novel chemoselective peptide bond forming reaction. SEA ligation is a useful reaction for protein total synthesis that is complementary to the native chemical ligation (NCL). In particular, SEA ligation proceeds efficiently in a wide range of pH, from neutral pH to pH 3-4. Thus, the pH can be chosen to optimize the solubility of the peptide segments or final product. It can be also chosen to facilitate the formation of difficult junctions, since the rate of SEA ligation increases significantly by decreasing the pH from 7.2 to 4.0. Here we describe a protocol for SEA ligation at pH 5.5 in the presence of 4-mercaptophenylacetic acid (MPAA) or at pH 4.0 in the presence of a newly developed diselenol catalyst. The protocols describe the formation of a valyl-cysteinyl peptide bond between two model peptides.<br>

scholarly journals A straightforward methodology to overcome solubility challenges for N-terminal cysteinyl peptide segments used in native chemical ligation

2021 ◽  
Author(s):  
Skander A. Abboud ◽  
El hadji Cisse ◽  
Michel Doudeau ◽  
Hélène Bénédetti ◽  
Vincent Aucagne
Keyword(s):  
Chemical Synthesis ◽  
Native Chemical Ligation ◽  
Chemical Ligation ◽  
Peptide Segments ◽  
Straightforward Approach

We herein describe a straightforward approach for the introduction of a solubilizing tag on N-terminal cysteinyl segments used in native chemical ligation-based protein chemical synthesis. Conveniently, the tag is removed during the ligation.

One-pot native chemical ligation by combination of two orthogonal thioester precursors

2017 ◽  
Vol 53 (13) ◽  
pp. 2114-2117 ◽  
Author(s):  
Yuya Asahina ◽  
Toru Kawakami ◽  
Hironobu Hojo
Keyword(s):  
Native Chemical Ligation ◽  
Protecting Group ◽  
One Pot ◽  
Chemical Ligation ◽  
Ligation Reaction ◽  
Ligation Method ◽  
Peptide Ligation

We developed a one-pot peptide ligation method using two orthogonal thioester precursors and a protecting group for the ligation reaction between Asp and Cys.

scholarly journals A Straightforward Methodology to Overcome Solubility Challenges for N-Terminal Cysteinyl Peptide Segments Used in Native Chemical Ligation

2020 ◽  
Author(s):  
Skander Abboud ◽  
El hadji Cisse ◽  
Michel Doudeau ◽  
Hélène Bénédetti ◽  
Vincent AUCAGNE
Keyword(s):  
Amino Acids ◽  
Chemical Synthesis ◽  
Building Blocks ◽  
Native Chemical Ligation ◽  
Synthetic Approach ◽  
Chemical Ligation ◽  
Limited Solubility ◽  
Peptide Segments

One of the main limitations encountered during the chemical synthesis of proteins through native chemical ligation (NCL) is the limited solubility of some of the peptide segments. The most commonly used solution to overcome this problem is to derivatize the segment with a temporary solubilizing tag. Conveniently, the tag can be introduced on the thioester segment in such a way that it is removed concomitantly with the NCL reaction. We herein describe a generalization of this approach to N-terminal cysteinyl segment counterparts, using a straightforward synthetic approach that can be easily automated from commercially available building blocks, and applied it to a well-known problematic target, SUMO-2 (93 amino acids).

scholarly journals Synthesis of protein kinase Cδ C1b domain by native chemical ligation methodology and characterization of its folding and ligand binding

2009 ◽  
Vol 15 (10) ◽  
pp. 642-646 ◽  
Author(s):  
Nami Ohashi ◽  
Wataru Nomura ◽  
Mai Kato ◽  
Tetsuo Narumi ◽  
Nancy E. Lewin ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Ligand Binding ◽  
Native Chemical Ligation ◽  
Chemical Ligation ◽  
Protein Kinase Cδ

scholarly journals SEA Ligation Is Accelerated at Mildly Acidic pH. Application to the Formation of Difficult Peptide Junctions

2019 ◽  
Author(s):  
Marine Cargoet ◽  
Vincent Diemer ◽  
Laurent Raibaut ◽  
Elizabeth Lissy ◽  
Benoît Snella ◽  
...  
Keyword(s):  
Total Synthesis ◽  
Peptide Bond ◽  
Native Chemical Ligation ◽  
Acidic Ph ◽  
Chemical Ligation ◽  
Bond Forming ◽  
Neutral Ph ◽  
Wide Range ◽  
Peptide Segments ◽  
Model Peptides

The bis(2-sulfanylethyl)amido (SEA)-mediated ligation has been introduced in 2010 as a novel chemoselective peptide bond forming reaction. SEA ligation is a useful reaction for protein total synthesis that is complementary to the native chemical ligation (NCL). In particular, SEA ligation proceeds efficiently in a wide range of pH, from neutral pH to pH 3-4. Thus, the pH can be chosen to optimize the solubility of the peptide segments or final product. It can be also chosen to facilitate the formation of difficult junctions, since the rate of SEA ligation increases significantly by decreasing the pH from 7.2 to 4.0. Here we describe a protocol for SEA ligation at pH 5.5 in the presence of 4-mercaptophenylacetic acid (MPAA) or at pH 4.0 in the presence of a newly developed diselenol catalyst. The protocols describe the formation of a valyl-cysteinyl peptide bond between two model peptides.<br>

scholarly journals The structure of SeviL, a GM1b/asialo-GM1 binding R-type lectin from the mussel Mytilisepta virgata

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kenichi Kamata ◽  
Kenji Mizutani ◽  
Katsuya Takahashi ◽  
Roberta Marchetti ◽  
Alba Silipo ◽  
...  
Keyword(s):  
Immune System ◽  
Sialic Acid ◽  
Ligand Binding ◽  
Steric Hindrance ◽  
Sequence Similarity ◽  
Binding Assays ◽  
Subunit Interface ◽  
Nmr Techniques ◽  
And Control ◽  
Asialo Gm1

AbstractSeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Ac$$\alpha$$ α (2-3)Gal$$\beta$$ β (1-3)GalNAc$$\beta$$ β (1-4)Gal$$\beta$$ β (1-4)Glc) and its precursor, asialo-GM1 (Gal$$\beta$$ β (1-3)GalNAc$$\beta$$ β (1-4)Gal$$\beta$$ β (1-4)Glc). The crystal structures of recombinant SeviL have been determined in the presence and absence of ligand. The protein belongs to the $$\beta$$ β -trefoil family, but shows only weak sequence similarity to known structures. SeviL forms a dimer in solution, with one binding site per subunit, close to the subunit interface. Molecular details of glycan recognition by SeviL in solution were analysed by ligand- and protein-based NMR techniques as well as ligand binding assays. SeviL shows no interaction with GM1 due to steric hindrance with the sialic acid branch that is absent from GM1b. This unusual specificity makes SeviL of great interest for the detection and control of certain cancer cells, and cells of the immune system, that display asialo-GM1.

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