Cation binding and conformation of human calmodulin-like protein.
The Ca(2+)-binding parameters of recombinant human calmodulin-like protein (CLP), a protein specifically expressed in mammary epithelial cells, were studied by flow dialysis in the absence and presence of 2, 10, and 30 mM MgCl2. In general, the four intrinsic binding constants (K'Ca) are about 8-fold lower than in animal and plant calmodulins. In the absence of Mg2+ the K'Ca values of the four binding steps equal 4.0 x 10(3), 3.3 x 10(4), 1.0 x 10(4), and 6.0 x 10(3) M-1, respectively. They allow us to distinguish two pairs of sites: a higher affinity pair with strong positive cooperativity and a lower affinity pair composed of non-interacting sites with different affinities. Mg2+ antagonizes Ca2+ binding by decreasing only Ca(2+)-binding steps 2 and 3, so that at high Mg2+ concentrations the positive cooperativity in the high-affinity pair has been lost and that the four K'Ca values are very similar with a mean K'Ca of 4 x 10(3) M-1. Direct Mg2+ binding studies by equilibrium gel filtration indicate that 4-5 Mg2+ bind to CLP with a mean K'Mg of 250 M-1. Conformational changes in the unique Tyr138 microenvironment, monitored by fluorimetry and near-UV difference spectrophotometry, indicate that in metal-free CLP this Tyr is shielded from the polar solvent and strongly quenched by a specific chemical group; Ca2+ binding induces a shift of Tyr to a more polar environment and removal of the quenching group, but without full exposure to the solvent.(ABSTRACT TRUNCATED AT 400 WORDS)
Durussel
I
I
Department of Biochemistry, University of Geneva, Switzerland.
Rhyner
J A
JA
Strehler
E E
EE
Cox
J A
JA
D000494:Allosteric Regulation; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008956:Models, Chemical; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013057:Spectrum Analysis; D014443:Tyrosine / Q000737:chemistry
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D011994:Recombinant Proteins; D014443:Tyrosine; D008274:Magnesium; D003545:Cysteine; D002118:Calcium