The CXXC motif at the N terminus of an α‐helical peptide

• Published in: Protein science 2006-08, Vol.15 (8), p.1945-1950
• Main Authors: Iqbalsyah, Teuku M., Moutevelis, Efrosini, Warwicker, Jim, Errington, Neil, Doig, Andrew J.
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Circular Dichroism; Computer Simulation; CXXC; Cysteine - chemistry; Dithiothreitol - chemistry; helix‐coil theory; Humans; Hydrogen-Ion Concentration; N‐cap; Oxidation-Reduction; Protein Folding; protein stability; Protein Structure, Secondary; Thioredoxins - chemistry; α‐helix
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.062271506

An active site containing a CXXC motif is always found in the thiol‐disulphide oxidoreductase superfamily. A survey of crystal structures revealed that the CXXC motif had a very high local propensity (26.3 ± 6.2) for the N termini of α‐helices. A helical peptide with the sequence CAAC at the N terminus was synthesized to examine the helix‐stabilizing capacity of the CXXC motif. Circular dichroism was used to confirm the helical nature of the peptide and study behavior under titration with various species. With DTT, a redox potential of Eo = −230 mV was measured, indicating that the isolated peptide is reducing in nature and similar to native human thioredoxin. The pKa values of the individual Cys residues could not be separated in the titration of the reduced state, giving a single transition with an apparent pKa of 6.74 (±0.06). In the oxidized state, the N‐terminal pKa is 5.96 (±0.05). Analysis of results with the modified helix‐coil theory indicated that the disulfide bond stabilized the α‐helical structure by 0.5 kcal/mol. Reducing the disulfide destabilizes the helix by 0.9 kcal/mol.