Characterization of pH titration shifts for all the nonlabile proton resonances a protein by two-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 p2H range 1.5-9 with two-dimensional (2D) 1H NMR. 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.
Kohda
D
D
Department of Molecular Physiology, Tokyo Metropolitan Institute of Medical Science, Japan.
Sawada
T
T
Inagaki
F
F
D000595:Amino Acid Sequence; D000818:Animals; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008297:Male; D051379:Mice; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D013363:Submandibular Gland
D011506:Proteins; D004815:Epidermal Growth Factor