Site-specific mutants of oncomodulin. 1H NMR and optical stopped-flow studies of the effect on the metal binding properties of an Asp59----Glu59 substitution in the calcium-specific site.
High resolution 1H nuclear magnetic resonance spectroscopy and optical stopped-flow techniques have been used to study the metal binding properties of a site-specific mutant of bacterial recombinant oncomodulin in which glutamate has replaced a liganding aspartate at position 59 in the CD calcium-binding site. In particular we have followed the replacement of calcium by lutetium in bacterial recombinant oncomodulin and D59E oncomodulin to provide a measure of the protein's preferences for metal ions of different ionic radii. The result of the Asp----Glu substitution is to make the mutant oncomodulin more similar to rat parvalbumin in terms of its relative CD- and EF-domain affinities for lutetium(III), that is to increase its affinity for metal ions with smaller ionic radii. This finding supports the original hypothesis that the presence of Asp at sequence position 59 is an important factor in the reduced preference of the CD site of oncomodulin for smaller metals such as magnesium (Williams, T. C., Corson, D. C., Sykes, B. D., and MacManus, J. P. (1987) J. Biol. Chem. 262, 6248-6256). However, our studies show that both the CD and the EF sites are affected by this single residue substitution suggesting that many factors play a role in the metal binding affinity and interaction between the two sites.
Golden
L F
LF
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Corson
D C
DC
Sykes
B D
BD
Banville
D
D
MacManus
J P
JP
D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D002135:Calcium-Binding Proteins; D005971:Glutamates; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C034733:oncomodulin; D001224:Aspartic Acid; D018698:Glutamic Acid; D002118:Calcium