Terbium-binding properties of calsequestrin from skeletal muscle sarcoplasmic reticulum.
Calsequestrin (Mr = 40,000) is a calcium-binding protein (Kd = 1 mM, 50 sites/molecule) located within the terminal cisternae of the sarcoplasmic reticulum of skeletal muscle cells. The interaction of terbium, a calcium analog, with rabbit skeletal muscle calsequestrin was studied by fluorescence and circular dichroism spectroscopy. Direct measurement of terbium binding using a fluorescence assay for terbium revealed that calsequestrin bound approx. 30 mol of terbium per mol of protein with an affinity of approx. 7 microM. The fluorescence of terbium measured at 545 nm was enhanced dramatically upon binding to calsequestrin, reaching a maximum value at a terbium to protein ratio of 28. The excitation spectrum of protein-bound terbium and chemical modification studies revealed that energy transfer occurred between aromatic residues, including tryptophan and bound terbium. Terbium bound to calsequestrin could be removed by EGTA, or displaced by Ca2+ or La3+. In the presence of Ca2+ or La3+ terbium bound to calsequestrin with a higher apparent affinity and lower capacity. 0.1 M KCl or 5 mM MgCl2 had little effect on terbium binding. Terbium increased the intrinsic fluorescence of calsequestrin 2-fold, and increased the alpha-helical content of calsequestrin from 16 to 33%. Terbium binding induces the same conformational changes in calsequestrin as does calcium, confirming that terbium is a useful calcium analog in this system.
D000818:Animals; D001665:Binding Sites; D001974:Bromosuccinimide; D002118:Calcium / Q000494:pharmacology; D002155:Calsequestrin / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D002942:Circular Dichroism; D004735:Energy Transfer; D007811:Lanthanum / Q000494:pharmacology; D008163:Luminescent Measurements; D009124:Muscle Proteins / Q000378:metabolism*; D009132:Muscles / Q000032:analysis*; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013725:Terbium / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism
D002155:Calsequestrin; D009124:Muscle Proteins; D013725:Terbium; D007811:Lanthanum; D014364:Tryptophan; D001974:Bromosuccinimide; D002118:Calcium