Disulfide conformation and design at helix N-termini
| Autor: | Ramachandra M. Bhaskara, Senthil Kumar, Sudhir Thakurela, Mansi Gupta, C. Ramakrishnan, S Indu, Raghavan Varadarajan |
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| Rok vydání: | 2009 |
| Předmět: |
Protein Denaturation
Protein Folding Circular dichroism Amino Acid Motifs Molecular Sequence Data intrahelical disulfides Biochemistry Protein Structure Secondary Thioredoxins Protein structure Structural Biology torsion angle Insulin Denaturation (biochemistry) Cysteine Disulfides Databases Protein Molecular Biology Research Articles chemistry.chemical_classification redox activity chemical denaturation biology Circular Dichroism CXXC motifs Active site thermostability Amino acid MODIP Crystallography chemistry Mutagenesis Helix biology.protein Thermodynamics Protein folding Thioredoxin Peptides Oxidation-Reduction Research Article |
| Zdroj: | Proteins |
| ISSN: | 0887-3585 |
| DOI: | 10.1002/prot.22641 |
| Popis: | To understand structural and thermodynamic features of disulfides within an α‐helix, a non‐redundant dataset comprising of 5025 polypeptide chains containing 2311 disulfides was examined. Thirty‐five examples were found of intrahelical disulfides involving a CXXC motif between the N‐Cap and third helical positions. GLY and PRO were the most common amino acids at positions 1 and 2, respectively. The N‐Cap residue for disulfide bonded CXXC motifs had average (ϕ,ψ) values of (−112 ± 25.2°, 106 ± 25.4°). To further explore conformational requirements for intrahelical disulfides, CYS pairs were introduced at positions N‐Cap‐3; 1,4; 7,10 in two helices of an Escherichia coli thioredoxin mutant lacking its active site disulfide (nSS Trx). In both helices, disulfides formed spontaneously during purification only at positions N‐Cap‐3. Mutant stabilities were characterized by chemical denaturation studies (in both oxidized and reduced states) and differential scanning calorimetry (oxidized state only). All oxidized as well as reduced mutants were destabilized relative to nSS Trx. All mutants were redox active, but showed decreased activity relative to wild‐type thioredoxin. Such engineered disulfides can be used to probe helix start sites in proteins of unknown structure and to introduce redox activity into proteins. Conversely, a protein with CYS residues at positions N‐Cap and 3 of an α‐helix is likely to have redox activity. Proteins 2010. © 2009 Wiley‐Liss, Inc. |
| Databáze: | OpenAIRE |
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