We have used electron paramagnetic resonance (EPR) to probe the homo- and heterooligomeric interactions of reconstituted sarcoplasmic reticulum Ca-ATPase (SERCA) and its regulator phospholamban (PLB). SERCA is responsible for restoring calcium to the sarcoplasmic reticulum to allow muscle relaxation, whereas PLB inhibits cardiac SERCA unless phosphorylated at Ser16. To determine whether changes in protein association play essential roles in regulation, we detected the microsecond rotational diffusion of both proteins using saturation transfer EPR. Peptide synthesis was used to create a fully functional and monomeric PLB mutant with a spin label rigidly coupled to the backbone of the transmembrane helix, while SERCA was reacted with a Cys-specific spin label. Saturation transfer EPR revealed that sufficiently high lipid/protein ratios minimized self-association for both proteins. Under these dilute conditions, labeled PLB was substantially immobilized after co-reconstitution with unlabeled SERCA, reflecting their association to form the regulatory complex. Ser16 phosphorylation slightly increased this immobilization. Complementary measurements with labeled SERCA showed no change in mobility after co-reconstitution with unlabeled PLB, regardless of its phosphorylation state. We conclude that phosphorylating monomeric PLB can relieve SERCA inhibition without changes in the oligomeric states of these proteins, indicating a structural rearrangement within the heterodimeric regulatory complex.
|Original language||English (US)|
|Number of pages||9|
|State||Published - Sep 19 2012|
Bibliographical noteFunding Information:
This work was supported in part by National Institutes of Health grants No. GM27906 and No. AR057220 (to D.D.T.). Z.M.J. was supported by National Institutes of Health grants No. AR007612 and No. GM008700, J.E.M. was supported by National Institutes of Health Training grant No. AR007612, and K.D.T. was supported by a Predoctoral Fellowship from the American Heart Association (grant No. 0615710Z). J.E.M. was enrolled in the graduate program in Physics and Astronomy.