Carbon-deuterium vibrational probes of amino acid protonation state

The protonation state of titratable amino acid residues has profound effects on protein stability and function. Therefore, correctly determining the acid dissociation constant, pK(a), of charged residues under physiological conditions is an important challenge. The general utility of site-specific carbon-deuterium (C-D) vibrational probes as reporters of the protonation state of arginine, aspartic acid, glutamic acid, and lysine amino acid side chains was examined using density functional theory (DFT) calculations. Substantial shifts were observed in the anharmonic vibrational frequencies of a C-D(2) probe placed immediately adjacent to the titratable group. Lysine exhibited the largest C-D(2) frequency shifts upon protonation, 44.9 cm(-1) (symmetric stretch) and 69.5 cm(-1) (asymmetric stretch). Furthermore, the predicted harmonic intensities of the C-D(2) probe vibrations were extraordinarily sensitive to the protonation state of the nearby acidic or basic group. Accounting for this dramatic change in intensity is essential to the interpretation of an infrared (IR) absorption spectrum that contains the signature of both the neutral and charged states.