Protein-protein interactions in calcium transport regulation probed by saturation transfer electron paramagnetic resonance.

摘要: We have used electron paramagnetic resonance (EPR) to probe the homo- and heterooligomeric interactions of reconstituted sarcoplasmic reticulum Ca-ATPase (SERCA) its regulator phospholamban (PLB). SERCA is responsible for restoring calcium allow muscle relaxation, whereas PLB inhibits cardiac unless phosphorylated at Ser16. To determine whether changes in protein association play essential roles regulation, we detected microsecond rotational diffusion both proteins using saturation transfer EPR. Peptide synthesis was create a fully functional monomeric mutant with spin label rigidly coupled backbone transmembrane helix, while reacted Cys-specific label. Saturation EPR revealed that sufficiently high lipid/protein ratios minimized self-association proteins. Under these dilute conditions, labeled substantially immobilized after co-reconstitution unlabeled SERCA, reflecting their form regulatory complex. Ser16 phosphorylation slightly increased this immobilization. Complementary measurements showed no change mobility PLB, regardless state. conclude phosphorylating can relieve inhibition without oligomeric states proteins, indicating structural rearrangement within heterodimeric