Author: Indu, S.; Kumar, Senthil T.; Thakurela, Sudhir; Gupta, Mansi; Bhaskara, Ramachandra M.; Ramakrishnan, C.; Varadarajan, Raghavan
Title: Disulfide conformation and design at helix Nâ€termini Cord-id: jqzb67xz Document date: 2009_10_20
ID: jqzb67xz
Snippet: To understand structural and thermodynamic features of disulfides within an αâ€helix, a nonâ€redundant dataset comprising of 5025 polypeptide chains containing 2311 disulfides was examined. Thirtyâ€five examples were found of intrahelical disulfides involving a CXXC motif between the Nâ€Cap and third helical positions. GLY and PRO were the most common amino acids at positions 1 and 2, respectively. The Nâ€Cap residue for disulfide bonded CXXC motifs had average (Ï•,ψ) values of (−112 ±
Document: To understand structural and thermodynamic features of disulfides within an αâ€helix, a nonâ€redundant dataset comprising of 5025 polypeptide chains containing 2311 disulfides was examined. Thirtyâ€five examples were found of intrahelical disulfides involving a CXXC motif between the Nâ€Cap and third helical positions. GLY and PRO were the most common amino acids at positions 1 and 2, respectively. The Nâ€Cap residue for disulfide bonded CXXC motifs had average (Ï•,ψ) values of (−112 ± 25.2°, 106 ± 25.4°). To further explore conformational requirements for intrahelical disulfides, CYS pairs were introduced at positions Nâ€Capâ€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 Nâ€Capâ€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 wildâ€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 Nâ€Cap and 3 of an αâ€helix is likely to have redox activity. Proteins 2010. © 2009 Wileyâ€Liss, Inc.
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