CHARACTERIZATION OF PH TITRATION SHIFTS FOR ALL THE NONLABILE PROTON RESONANCES IN A PROTEIN BY 2-DIMENSIONAL NMR - THE CASE OF MOUSE EPIDERMAL GROWTH-FACTOR

The pH titration shifts for all the nonlabile proton resonances in a 53-residue protein (mouse epidermal growth factor) were measured in the pH-2 range 1.5-9 with two-dimensional (2D) H-1NMR. The 2D NMR pH titration experiment made it possible to determine the pK values for all the ionizable groups which were titrated in the pH range 1.5-9 in the protein. The pK values of the nine ionizable groups (alpha-amino group, four Asp, two Glu, one His, and alpha-carboxyl group) were found to be near their normal values. The 2D titration experiment also provided a detailed description of the pH-dependent behavior of the proton chemical shifts and enabled us to characterize the pH-dependent changes of protein conformation. Analysis of the pH-dependent shifts of ca. 200 proton resonances offered evidence of conformational changes in slightly basic pH solution: The deprotonation of the N-terminal alpha-amino group induced a widespread conformational change over the beta-sheet structure in the protein, while the effects of deprotonation of the His22 imidazole group were relatively localized. We found that the 2D NMR pH titration experiment is a powerful tool for investigating the structural and dynamic properties of proteins.