Indu, S and Kumar, Senthil T and Thakurela, Sudhir and Gupta, Mansi and Bhaskara, Ramachandra M and Ramakrishnan, C and Varadarajan, Raghavan (2010) Disulfide conformation and design at helix N-termini. In: Proteins: Structure, Function, and Genetics, 78 (5). pp. 1228-1242.
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To understand structural and thermodynamic features of disulfides within an alpha-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 values of (-112 +/- 25.2 degrees, 106 +/- 25.4 degrees). 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 alpha-helix is likely to have redox activity.
|Item Type:||Journal Article|
|Additional Information:||Copyright of this article belongs to John Wiley and Sons.|
|Keywords:||intrahelical disulfides; CXXC motifs; MODIP; torsion angle; chemical denaturation; thermostability; redox activity|
|Department/Centre:||Division of Biological Sciences > Molecular Biophysics Unit|
|Date Deposited:||11 Jun 2010 04:19|
|Last Modified:||19 Sep 2010 05:59|
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