IONIZATION OF CYSTEINE RESIDUES AT THE TERMINI OF MODEL ALPHA-HELICAL PEPTIDES - RELEVANCE TO UNUSUAL THIOL PK(A) VALUES IN PROTEINS OF THE THIOREDOXIN 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 lower 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 helix 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 absorbance at 240 nm, of the alpha-helix content measured by the mean residue ellipticity at 222 nm, and of the chemical shifts of protons close to the sulphur atom of the cysteine residue. The favourable interaction 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, corresponding to a stabilisation energy of 2.1 kcal/mol. At the C terminus, 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 dipole with the cysteine thiolate anion, involving both its charge and hydrogen-bonding. Subtle conformational effects in different model peptides appear to influence the ionisation of the thiol group significantly, with an N terminal Cys-Pro sequence having the most favourable interaction with the alpha-helix. (C) 1995 Academic Press Limited