IONIZATION OF CYSTEINE RESIDUES AT THE TERMINI OF MODEL ALPHA-HELICALPEPTIDES - RELEVANCE TO UNUSUAL THIOL PK(A) VALUES IN PROTEINS OF THETHIOREDOXIN FAMILY

The physical basis of the unusually low pK(a) values of an active site cysteine thiol group in proteins with the thioredoxin fold is unknown . The electrostatic field associated with an alpha-helix pointing with its N terminus towards the cysteine residue has been implicated to lo wer the thiol pK(a) value by up to 5 pH units in glutaredoxin and DsbA . Here, the influence of the presence of an alpha-helical conformation on the ionisation of a cysteine thiol group located at or near the he lix terminus is investigated in highly helical synthetic peptides with the generic sequence Ac-AAAAAAAAARAAAARAAAARAA-(NH2). The thiol pK(a) values have been determined by monitoring the pH dependence of the ab sorbance at 240 nm, of the alpha-helix content measured by the mean re sidue ellipticity at 222 nm, and of the chemical shifts of protons clo se to the sulphur atom of the cysteine residue. The favourable interac tion between the thiolate anion at the N terminus and the alpha-helix decreases the thiol pK(a) value by up to 1.6 pH units when compared to a normal thiol pK(a) value measured in an unfolded control peptide, c orresponding to a stabilisation energy of 2.1 kcal/mol. At the C termi nus, the thiol pK(a) value is increased, but by only 0.2 pH units. The observations are consistent with an interaction of the alpha-helix di pole with the cysteine thiolate anion, involving both its charge and h ydrogen-bonding. Subtle conformational effects in different model pept ides appear to influence the ionisation of the thiol group significant ly, with an N terminal Cys-Pro sequence having the most favourable int eraction with the alpha-helix. (C) 1995 Academic Press Limited