1H-n.m.r. study of the folding of ribonuclease 12-(β-(3-pyridyl)-l-Ala) S-peptide (1–14)

The ¹H-n.m.r. spectra (360 MHz) of 12-(β-(3-pyridyl)-l -Ala) ribonuclease S-peptide (1–14), a tetradecapeptide incorporating (β-3-pyridyl-l -Ala) instead of His at position 12, have been assigned. The shift vs. temperature dependence has been analyzed at three different pD's in terms of a two-state helix (3–13) ± coil equilibrium, and the corresponding values for the thermodynamic quantities ΔH° and ΔS° determined. Helix populations at 0°C have been measured as a function of pD, showing their dependence on two apparent pK_a's at ? 3.3 and 5.5, with a maximum at pD ? 4.2. All the obtained results show that the new peptide has very similar folding properties to those shown by S-peptide and particularly to those of C-peptide. The 3–13 helix formed is stabilized by two interactions: a salt-bridge Glu 2^-. Arg 10+ and a partial stacking between the aromatic rings of residues Phe 8 and His 12. Calculations involving ring current shifts and potential energies validate the possible existence of this latter interaction, which must present a local geometry defined by χ₁X⁸ 180°, χ₂X⁸ 100°, χ₁¹² – 60 and χ₂¹² 80.