13C NMR studies of 13C methylated Fc fragments

Little is known about the structure-function relationships in the Fc portion of the IgG molecule, even though it mediates a number of physiological effector functions. This study is focused on the characterization of the lysine residues because of their potential involvement in Fc effector interactions. The Fc fragments were reductively 13C-methylated and 13C NMR was used to characterize the dimethylamino groups. Dimethylamino groups have properties generally representative of native amino groups. In addition, the chemical shifts of the 13C NMR resonances are quite sensitive to the local environment of the dimethylamino moiety. All of the lysyl amino groups of the Fc fragments were modified as indicated by the relative areas of the dimethyllysyl and the N-terminal dimethylamino 13C NMR resonances. The pH dependences of the fragments were determined and seven unique titration curves were resolved for Fc-Cohn while eight curves were obtained for Fc-Tu. The calculated pKa values from these titration curves fell in the range of 9.6 to 10.3 for both fragments. Several of the titrating resonances are from single lysyl residues. Current efforts are directed toward assignment of the 13C NMR resonances to specific lysyl residues and toward further characterization of their thermodynamic properties