Spectral studies of the Ca2+-dependent interaction of trifluoperazine with S100b.
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+, the k1/2 value of TFP was 130 microM, while its k1/2 value decreased to 28 microM 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 x 10(2) 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.
Pingerelli
P L
PL
Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202.
Mizukami
H
H
Mooney
M J
MJ
Schlaepfer
A L
AL
D002135:Calcium-Binding Proteins / Q000378:metabolism*; D005461:Fluorine; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009414:Nerve Growth Factors; D011485:Protein Binding; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D014268:Trifluoperazine / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D009414:Nerve Growth Factors; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D014268:Trifluoperazine; D005461:Fluorine