Side chain flexibility and protonation states of sulfur atom containing amino acids

We present a set of new data allowing elucidation of the energetic, conformational and vibrational features of cysteine (Cys) and methionine (Met), i.e. two natural amino acids (AAs) containing a sulfur atom in their side chains. Special attention has been paid to cysteine, for which vibrational features were analysed in a wide pH range (6-to-12), where its backbone can switch from a zwitterionic to an anionic form, and its side chain SH group can be deprotonated. Through a detailed discussion on the relative acidity of the three protonation sites of this AA, as well as on the vibrational markers arising from zwitterionic and anionic backbones, we could assign the spectra recorded at pH 6, 9.2 and 12 to three species, referred to as Cys(0), Cys(1-)(a) and Cys(2-), where the superscripts designate their global net charges. To bring clarification to the structural and vibrational features, quantum mechanical calculations based on the Density Functional Theory (DFT) were carried out, allowing (i) a quasi exhaustive energetic and side chain conformational analysis through 804 clusters of explicitly hydrated AAs; (ii) simulation of the observed aqueous solution vibrational spectra of Cys(0), Cys(-2) and Met by means of the theoretical data obtained from their conformationally distinct lowest energy clusters.