Measurement of side-chain carboxyl pKa values of glutamate and aspartate residues in an unfolded protein by multinuclear NMR spectroscopy

A triple-resonance NMR pulse scheme is presented for measuring aspartic and glutamic acid side-chain pK(a) values in unfolded protein states where chemical shift overlap is limiting. The experiment correlates side-chain carboxyl carbon chemical shifts of these residues with the backbone amide proton chemical shift of the following residue. The methodology is applied to an N-15, C-13 labeled sample of the N-terminal SH3 domain of the Drosophila protein drk, which exists in equilibrium between folded (Fe c,) and unfolded (U-exch) states under nondenaturing conditions. Residue-specific pK(a) values of side-chain carboxyl groups are presented for the first time for an unfolded protein (drk U-exch state), determined from a pH titration. Results indicate that deviations from pK(a) values measured for model compounds are likely due to local effects, while long-range electrostatic interactions appear to be of minor importance for this protein.