The binding of holmium(III) to bovine brain calmodulin (spin labeled at tyrosine-99) was examined by electron paramagnetic resonance (EPR) spectroscopy. Changes in the EPR spectrum detected during the titration of the spin-labeled calmodulin with HoIII indicate that HoIII binds initially to the carboxyl terminal domain of the protein, which contains two of the four calcium ion binding sites. The EPR spectral changes associated with the binding of the first two HoIII ions reflect a significant decrease in the mobility of the nitroxide spin label; however, subsequent HoIII addition results in EPR spectra reflective of increased motion. The increased motion may well be associated with the major conformational change (i.e. from "globular" to "dumb-bell" shape) known to occur following the binding of two "activating" metal ions to the protein.
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