S. Indu and Senthil T. Kumar contributed equally to this work.
Disulfide conformation and design at helix N-termini
Article first published online: 20 OCT 2009
Copyright © 2009 Wiley-Liss, Inc.
Proteins: Structure, Function, and Bioinformatics
Volume 78, Issue 5, pages 1228–1242, April 2010
How to Cite
Indu, S., Kumar, S. T., Thakurela, S., Gupta, M., Bhaskara, R. M., Ramakrishnan, C. and Varadarajan, R. (2010), Disulfide conformation and design at helix N-termini. Proteins, 78: 1228–1242. doi: 10.1002/prot.22641
- Issue published online: 8 FEB 2010
- Article first published online: 20 OCT 2009
- Accepted manuscript online: 20 OCT 2009 12:00AM EST
- Manuscript Accepted: 13 OCT 2009
- Manuscript Revised: 26 SEP 2009
- Manuscript Received: 10 JUL 2009
- Council of Scientific and Industrial Research, Government of India
- Department of Biotechnology, Government of India
- intrahelical disulfides;
- CXXC motifs;
- torsion angle;
- chemical denaturation;
- redox activity
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.