Probing the metal-binding sites of cod parvalbumin using europium(III) ion luminescence and diffusion-enhanced energy transfer.
Excitation spectroscopy of the 7F0----5D0 transition of Eu3+ and diffusion-enhanced energy transfer are used to study metal-binding characteristics of the calcium-binding protein parvalbumin from codfish. Energy is transferred from Eu3+ ions occupying the CD- and EF-binding sites to the freely-diffusing Co(III) coordination complex energy acceptors: [Co(NH3)6]3+, [Co(NH3)5H2O]3+, [CoF(NH3)5]2+, [CoCl(NH3)5]2+, [Co(NO2)3(NH3)3], and [Co(ox)3]3-. In the absence of these inorganic energy acceptors, the excited-state lifetimes of Eu3+ bound to the CD and EF sites are indistinguishable, even in D2O; however, in the presence of the positively charged energy acceptor complexes, the Eu3+ probes in the cod parvalbumin have different excited-state lifetimes due to a greater energy-transfer site from Eu3+ in the CD site than from this ion in the EF site. The observation of distinct lifetimes for Eu3+ in the two sites allows the study of the relative binding site affinities and selectivity, using other members of the lanthanide ion series. Our results indicate that during the course of a titration of the metal-free protein, Eu3+ fills the two sites simultaneously. Eu3+ is competitively displaced by other Ln3+ ions, with the CD site showing a preference for the larger Ln3+ ions while the EF site shows little, if any, competitive selectivity across the Ln3+ ion series.
Cronce
D T
DT
Department of Chemistry, Pennsylvania State University, University Park 16802.
Horrocks
W D
WD
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism; D004058:Diffusion; D004735:Energy Transfer; D005063:Europium / Q000378:metabolism*; D005399:Fishes; D007700:Kinetics; D007811:Lanthanum / Q000378:metabolism; D008163:Luminescent Measurements; D008670:Metals / Q000378:metabolism*; D008674:Metals, Rare Earth / Q000378:metabolism; D010320:Parvalbumins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013057:Spectrum Analysis
D008670:Metals; D008674:Metals, Rare Earth; D010320:Parvalbumins; D003035:Cobalt; D005063:Europium; D007811:Lanthanum; D002118:Calcium