Calcium binding properties of recombinant calcium binding protein 40, a major calcium binding protein of lower eukaryote Physarum polycephalum.
Calcium binding protein 40 (CBP40) is a Ca(2+)-binding protein abundant in the plasmodia of Physarum polycephalum. CBP40 consists four EF-hand domains in the COOH-terminal half and a putative alpha-helix domain in the NH(2)-terminal half. We expressed recombinant proteins of CBP40 in Escherichia coli to investigate its Ca(2+)-binding properties. Recombinant proteins of CBP40 bound 4 mol of Ca(2+) with much higher affinity (pCa(1/2) = 6.5) than that of calmodulin. When residues 1-196 of the alpha-helix domain were deleted, the affinity for Ca(2+) decreased to pCa(1/2) = 4.6. A chimeric calmodulin was generated by conjugating the alpha-helix domain of CBP40 with calmodulin. The affinity of Ca(2+) for the chimeric calmodulin was higher than that for calmodulin, suggesting that the alpha-helix domain is responsible for the high affinity of CBP40 for Ca(2+). CBP40 forms large aggregates reversibly in a Ca(2+)-dependent manner. A mutant protein with a deletion of NH(2)-terminal 32 residues, however, could not aggregate, indicating the importance of these residues for the aggregation. The aggregation occurs above micromolar levels of Ca(2+) concentration, so it may only occur when CBP40 is secreted out of the plasmodial cells.
Nakamura
A
A
Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan.
Okagaki
T
T
Takagi
T
T
Nakashima
K i
Ki
Yazawa
M
M
Kohama
K
K
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020832:EF Hand Motifs / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010804:Physarum / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism
C406924:CBP40 protein, Physarum; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D010446:Peptide Fragments; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D002118:Calcium