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

被引:29
作者
KOHDA, D [1 ]
SAWADA, T [1 ]
INAGAKI, F [1 ]
机构
[1] TOKYO METROPOLITAN INST MED SCI,DEPT MOLEC PHYSIOL,18-22 HONKOMAGOME 3 CHOME,BUNKYO KU,TOKYO 113,JAPAN
关键词
D O I
10.1021/bi00234a009
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:4896 / 4900
页数:5
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