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Synthesis and Structure Determination of µ-Conotoxin PIIIA Isomers with Different Disulfide Connectivities
Published on: October 2, 2018
Disulfide conformation and design at helix N-termini
S Indu1, Senthil T Kumar, Sudhir Thakurela
1Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Proteins
|November 26, 2009
Summary
Intrahelical disulfides, particularly CXXC motifs at alpha-helix N-Cap-3 positions, can form spontaneously. Engineered disulfides can probe helix start sites and introduce redox activity.
Area of Science:
- Protein structure and biophysics
- Biochemistry and molecular biology
Background:
- Disulfide bonds are crucial for protein structure and function.
- Alpha-helices are common secondary structures in proteins.
Purpose of the Study:
- To investigate the structural and thermodynamic properties of disulfides within alpha-helices.
- To explore the conformational requirements for intrahelical disulfide formation.
- To engineer novel disulfide bonds for protein structure probing and redox activity introduction.
Main Methods:
- Analysis of a non-redundant protein dataset containing 5025 polypeptide chains and 2311 disulfides.
- Site-directed mutagenesis to introduce cysteine pairs at specific positions in Escherichia coli thioredoxin.
- Chemical denaturation and differential scanning calorimetry to assess mutant stability.
- Redox activity assays.
Main Results:
- Identified 35 instances of intrahelical disulfides involving a CXXC motif at N-Cap-3 positions.
- Disulfides formed spontaneously only at N-Cap-3 positions in engineered thioredoxin.
- All engineered mutants were destabilized and exhibited decreased redox activity compared to wild-type.
Conclusions:
- The N-Cap-3 position is favorable for spontaneous intrahelical disulfide formation.
- Engineered intrahelical disulfides can serve as structural probes and introduce redox functionality.
- Proteins with cysteine residues at N-Cap and 3 of an alpha-helix are likely redox active.
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