Purification and Characterization of the Recombinant Human Calcium-Binding S100 Proteins CAPL and CACY

Abstract

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.

Binding of Ca2+ and Zn2+ to Human Nuclear S100A2 and Mutant Proteins

“…8). These results were confirmed in more recent studies on S100A4, S100A6, S100B, and S100A11 (25)(26)(27). Zn 2ϩ can affect the binding of Ca 2ϩ to particular S100 proteins (28 -30).…”

“…Underlined are the inserted restriction sites for BamHI, EcoRI, SalI, and PstI, and in italics is the sequence of the EF-site of PV. The resulting proteins are N mutant (change of positions [21][22][23][24][25][26][27][28][29][30][31][32][33][34] and NC mutant (change of positions 21-34 and 64 -75).…”

“…Expression and Purification of Mutant Proteins-Recombinant S100A2 proteins were expressed and purified as described previously (25). For the NC mutant, the purification procedure was modified in that the supernatant applied to the phenyl-Sepharose affinity column contained only 1 mM CaCl 2 , and proteins were eluted with 20 mM Tris-HCl (pH 7.5) containing 10 mM EGTA.…”

“…Proteins were visualized by Coomassie Blue or silver staining. The molecular mass of the purified recombinant S100A2 proteins was analyzed by electrospray ionization mass spectrometry as described (25).…”

“…Zn 2ϩ can affect the binding of Ca 2ϩ to particular S100 proteins (28 -30). Indeed, using direct binding methods for S100B (27,28), S100A4 and S100A6 (25), S100A3 (31), S100A12 (32), and S100A9 (33), it was demonstrated that they are able to bind Zn 2ϩ with fairly high affinity. Thus, S100 proteins have variable cation binding properties in agreement with their diversified functions.…”