title: Disulfide conformation and design at helix N-termini.
abstract: 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 (phi,psi) 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.
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
authors: S
authors: S
authors: Indu
authors: Senthil T
authors: ST
authors: Kumar
authors: Sudhir
authors: S
authors: Thakurela
authors: Mansi
authors: M
authors: Gupta
authors: Ramachandra M
authors: RM
authors: Bhaskara
authors: C
authors: C
authors: Ramakrishnan
authors: Raghavan
authors: R
authors: Varadarajan
mesh_terms: D020816:Amino Acid Motifs; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D030562:Databases, Protein; D004220:Disulfides / Q000737:chemistry*; D007328:Insulin / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics
chemical_list: D004220:Disulfides; D007328:Insulin; D010455:Peptides; D013879:Thioredoxins; D003545:Cysteine