Abstract
Abstract The circular dichroism spectra of calsequestrin and the high affinity calcium-binding protein from rabbit skeletal muscle sarcoplasmic reticulum have been recorded in the presence and absence of Ca2+ and other cations. Calsequestrin Forms I and II show decreases in negativity upon Ca2+ binding from -12 to -6 x 103 and -20 to -10 x 103 deg cm2 dmole-1, respectively, at 200 nm, and increases from -5 to -6 x 103 and -7 to -10 x 103 deg cm2 dmole-1 at 220 nm. Above 250 nm there is little optical activity in the absence of cations, but both forms of calsequestrin show a complex signal with maximum of about 90 deg cm2 dmole-1 between 270 and 285 nm in their presence. The changes in circular dichroism are interpreted as reflecting a large conformational change in the protein upon cation binding. Absorption difference spectra in the 320 to 240 nm range confirm that calcium binding induces perturbation in the absorption bands of tryosine, tryptophan, and phenylalanine, probably as a result of greater folding. The circular dichroism and absorption spectra of the high affinity calcium-binding protein do not change upon binding calcium. This protein, even in the absence of calcium, has intense circular dichroism activity in the aromatic region, with a maximum of 250 deg cm2 dmole-1 at about 280 nm.
Highlights
The circular dichroism spectra of calsequestrin and the high affinity calcium-binding protein from rabbit skeletal muscle sarcoplasmic reticulum have been recorded in the presence and absence of Ca2+ and other cations
The circular dichroism and absorption spectra of the high affinity calcium-binding protein do not change upon binding calcium
When the CD spectrum of calsequestrin Form I (8) was recorded in the absence of added Ca2+, in the region between 250 and 190 nm, a spectrum with an intense negative band at 200 nm and a poorly resolved shoulder near 220 nm was seen (Fig. 1)
Summary
Proteirhs-Calsequestrin (8) and the high affinity calcium-binding protein (5) were isolated from rabbit sarcoplasmic reticulum as previously described.Circular Dichroism-CD spectra were recorded using an ORD/CD-15 spectropolarimeter (Durruu-Janan Snectroscooic Co.)equipped- with-the SS-20 Cb modification (iproule &ientific; Boulder Creek). Proteirhs-Calsequestrin (8) and the high affinity calcium-binding protein (5) were isolated from rabbit sarcoplasmic reticulum as previously described. Circular Dichroism-CD spectra were recorded using an ORD/. Equipped- with-the SS-20 Cb modification (iproule &ientific; Boulder Creek). Spectra were analyzed between 320 and 250 nm with protein concentrations ranging from 0.5 to 1.0 mg per ml in a rectangular cell of 1.0.cm path length. Spectra were analyzed below 250 nm with a protein concentration between 0.05 and 0.1 mg per ml and a cell path length 0.10 cm. Spectra were recorded using a time constant of 16 s and scanning speeds of 1.1 to 3.6 nm per min. The variability & repeated scans for a typical
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