Characterization of pH Titration Shifts for All the Nonlabile Proton Resonances in a Protein by Two-Dimensional NMR: The Case of Mouse Epidermal Growth Factor

Daisuke Kohda, Toshie Sawada, Fuyuhiko Inagaki

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

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 (α-amino group, four Asp, two Glu, one His, and α-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 α-amino group induced a widespread conformational change over the β-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.

Original languageEnglish
Pages (from-to)4896-4900
Number of pages5
JournalBiochemistry
Volume30
Issue number20
DOIs
Publication statusPublished - May 1 1991

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Titration
Epidermal Growth Factor
Protons
Nuclear magnetic resonance
Deprotonation
Proteins
Experiments
Chemical shift
Conformations
Protein Conformation
Reference Values

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Characterization of pH Titration Shifts for All the Nonlabile Proton Resonances in a Protein by Two-Dimensional NMR : The Case of Mouse Epidermal Growth Factor. / Kohda, Daisuke; Sawada, Toshie; Inagaki, Fuyuhiko.

In: Biochemistry, Vol. 30, No. 20, 01.05.1991, p. 4896-4900.

Research output: Contribution to journalArticle

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