Purification and characterization of the recombinant human calcium-binding S100 proteins CAPL and CACY.
The S100 proteins CAPL and CACY are expressed in a tissue- and cell-specific manner and have been reported to be associated with the metastatic phenotype of tumor cells. In order to study the biochemical, cation-binding, and conformational properties, we produced and purified large amounts of the recombinant human proteins in Escherichia coli. Several characteristics of native proteins are shown to correspond to those of the bacterially expressed proteins. Both are able to form homodimers in vitro, probably the biologically active species, but not heterodimers. The Ca(2+)-binding parameters were studied by flow offlysis at physiological ionic strength. Both isotherms show a maximum of two Ca2+ per protein and are insensitive to Mg2+, indicating that the sites are of the Ca(2+)-specific type. The isotherms show slight (CAPL, nH = 1.15) or pronounced (CACY, nH = 1.33) positive cooperativity with K0.5 values of 0.32 mM (CACY) and 0.15 mM (CAPL), indicating that the sites are of the low-affinity type. Conformational changes in the Tyr microenvironment of CACY indicate that Ca2+ binding induces a shift of Tyr to a less polar environment. Mg2+ does not affect the fluorescence properties nor does it induce a difference spectrum, thus suggesting that at physiological ionic conditions it does not interact with the protein. The Ca(2+)-induced difference spectra of CAPL are about 3 times smaller than those of CACY, suggesting that the additional Tyr84 in CACY is much more sensitive to Ca2+ than the two Tyr residues conserved in both proteins.
Pedrocchi
M
M
Department of Pediatrics, University of Zürich, Switzerland.
Schäfer
B W
BW
Durussel
I
I
Cox
J A
JA
Heizmann
C W
CW
D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018797:Cell Cycle Proteins; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000075383:S100 Calcium Binding Protein A6; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D000075383:S100 Calcium Binding Protein A6; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C499412:S100a4 protein, rat; C489132:S100a6 protein, rat; C055351:S100A6 protein, human; C077591:S100A4 protein, human