Measurement of Side-Chain Carboxyl pKaValues of Glutamate and Aspartate Residues in an Unfolded Protein by Multinuclear NMR Spectroscopy

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Abstract
A triple-resonance NMR pulse scheme is presented for measuring aspartic and glutamic acid side-chain pKa 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 15N, 13C labeled sample of the N-terminal SH3 domain of the Drosophila protein drk, which exists in equilibrium between folded (Fexch) and unfolded (Uexch) states under nondenaturing conditions. Residue-specific pKa values of side-chain carboxyl groups are presented for the first time for an unfolded protein (drk Uexch state), determined from a pH titration. Results indicate that deviations from pKa 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.