Depolymerization of phospholamban in the presence of calcium pump: a fluorescence energy transfer study.
Phospholamban (PLB), a 52-amino acid protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum (SR) through PLB phosphorylation mediated by beta-adrenergic stimulation. The mobility of PLB on SDS-PAGE indicates a homopentamer, and it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, the oligomeric structure of PLB must be determined in its native milieu, a lipid bilayer containing the Ca-ATPase. Here we have used fluorescence energy transfer (FET) to study the oligomeric structure of PLB in SDS and dioleoylphosphatidylcholine (DOPC) lipid bilayers reconstituted in the absence and presence of Ca-ATPase. PLB was labeled, specifically at Lys 3 in the cytoplasmic domain, with amine-reactive fluorescent donor/acceptor pairs. FET between donor- and acceptor-labeled subunits of PLB in SDS solution and DOPC lipid bilayers indicated the presence of PLB oligomers. The dependence of FET efficiency on the fraction of acceptor-labeled PLB in DOPC bilayers indicated that it is predominantly an oligomer having 9-11 subunits, with approximately 10% of the PLB as monomer, and the distance between dyes on adjacent PLB subunits is 0.9 +/- 0.1 nm. When labeled PLB was reconstituted with purified Ca-ATPase, FET indicated the depolymerization of PLB into smaller oligomers having an average of 5 subunits, with a concomitant increase in the fraction of monomer to 30-40% and a doubling of the intersubunit distance. We conclude that PLB exists primarily as an oligomer in membranes, and the Ca-ATPase affects the structure of this oligomer, but the Ca-ATPase binds preferentially to the monomer and/or small oligomers. These results suggest that the active inhibitory species of PLB is a monomer or an oligomer having fewer than 5 subunits.
Reddy
L G
LG
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455, USA.
Jones
L R
LR
Thomas
D D
DD
D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D003902:Detergents; D004735:Energy Transfer; D005456:Fluorescent Dyes; D005960:Glucosides; D008956:Models, Chemical; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010713:Phosphatidylcholines; D011108:Polymers / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D013050:Spectrometry, Fluorescence; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D003902:Detergents; D005456:Fluorescent Dyes; D005960:Glucosides; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D011108:Polymers; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D000099211:Phospholamban; C018619:octyl-beta-D-glucoside; C017251:1,2-oleoylphosphatidylcholine