Investigating site-specific effects of the -X glutamate in a parvalbumin CD site model peptide.
The -X glutamate in a 33-residue model peptide comprising the CD site of carp parvalbumin 4.25 (ParvCD) was replaced with aspartate (ParvCD-XD) and the effect on calcium-dependent dimerization and calcium affinity assessed. The peptide ParvCD demonstrates a 10(5)-fold lower calcium affinity than the same site in the native protein. Both the ParvCD and ParvCD-XD model peptides fail to bind magnesium. The low calcium affinity and failure of the model ParvCD site to bind magnesium may be due to higher enthalpic costs of chelation by the -X glutamate. Replacement of the -X glutamate with an aspartate resulted in a twofold increase in the calcium affinity of both the monomer and dimer forms and a twofold increase in the calcium dependent dimerization of the peptide. A -X glutamate to aspartate replacement in 33-residue model peptides corresponding to bovine brain calmodulin site 3 (R. M. Procyshyn and R. E. Reid, Arch. Biochem. Biophys. 311, 425-429, 1994) and in Escherichia coli d-galactose-binding protein (S. K. Drake, K. L. Lee, and J. J. Falke, Biochemistry 35, 6697-6705, 1996) agree with results in the ParvCD site. However, in rat oncomodulin a -X glutamate to aspartate replacement increases calcium affinity (R. C. Hapak, P. J. Lammers, W. A. Palmisano, E. R. Birnbaum, and M. T. Henzl, J. Biol. Chem. 264, 18751-18760, 1989). The different effect of a -X glutamate to aspartate substitution in the different sites suggests site-specific factors dictating the thermodynamic contribution of the -X glutamate to calcium affinity.
Franchini
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PL
Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, British Columbia, V6T 1Z3, Canada.
Reid
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RE
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002347:Carps; D002417:Cattle; D002942:Circular Dichroism; D019281:Dimerization; D018698:Glutamic Acid / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010320:Parvalbumins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D051381:Rats; D013816:Thermodynamics
D010320:Parvalbumins; D010446:Peptide Fragments; D001224:Aspartic Acid; D018698:Glutamic Acid; D002118:Calcium