Electron spin resonance of calmodulin-vanadyl complexes.
X-band (9.2 GHz) electron spin resonance spectroscopy was used to investigate the binding of vanadyl to calmodulin. Solution spectra, obtained at ambient temperature with various VO2+:calmodulin molar ratios, suggested a binding stoichioimetry of 4 mol of VO2+/mol of protein and the possibility of two classes of binding sites. The latter was confirmed by using frozen solutions of calmodulin-VO2+ complexes that gave splitting of the spectral bands corresponding to the parallel components, which was particularly pronounced with the three high-field peaks. Competition of Ca2+ for the VO2+ binding sites was investigated, and the results indicated that two of the VO2+ sites corresponded to two of the Ca2+ sites; the other two VO2+ binding sites may have a higher affinity for VO2+ than for Ca2+ or they may correspond to Ca2+-independent sites. These results demonstrate that electron spin resonance spectroscopy can be used advantageously to probe subtle differences in the microenvironments of metal-binding sites in calmodulin.
Nieves
J
J
Department of Chemistry, University of Miami, Florida 33124.
Kim
L
L
Puett
D
D
Echegoyen
L
L
Benabe
J
J
Martinez-Maldonado
M
M
D001665:Binding Sites; D002147:Calmodulin; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D014638:Vanadates
D002147:Calmodulin; D014638:Vanadates