Spectral studies of the Ca2+-dependent interaction of trifluoperazine with S100b

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Abstract
S100b is a calcium-binding protein that will bind to many calmodulin target molecules in a Ca2+-dependent manner. In order to study the Ca2+-dependent binding properties of S100b, its interaction with a calmodulin antagonist, trifluoperazine (TFP), was investigated using [19F]- and [1H]-NMR and UV-difference spectroscopy. It was estimated from [19F]-NMR that in the absence of Ca2+, thek 1/2 value of TFP was 130 µM, while itsk 1/2 value decreased to 28 µM in the presence of Ca2+. The addition of KCl was not antagonistic to the Ca2+-dependent interaction of TFP to S100b. The chemical exchange rate of TFP with Ca2+-bound S100b was estimated to be 9×102 sec−1. By comparison with TFP-calmodulin exchange rates, it is suggested that the TFP-binding site on S100b is structurally different from its binding sites on calmodulin. Proton NMR resonance broadening in the range 6.8–7.2 ppm, corresponding to phenylalanine nuclei of S100b, indicates that these residues may be involved in TFP binding. Addition of Ca2+ to a 1:1 mixture of S100b and TFP resulted in a red-shifted UV-difference spectrum, while no significant difference spectrum was detected when Mg2+ was added to a S100b-TFP solution. Thus, we suggest that Ca2+ induces the exposure of a hydrophobic domain on S100b containing one or more phenylalanine residues that will bind TFP but that this domain is different from the hydrophobic domain on calmodulin.