Sarcolipin, the shorter homologue of phospholamban, forms oligomeric structures in detergent micelles and in liposomes

The human 31-amino acid integral membrane protein sarcolipin (SLN), which r egulates the sarcoplasmic reticulum Ca-ATPase in fast-twitch skeletal muscl e, was chemically synthesized. Appropriate synthesis and purification strat egies were used to achieve high purity and satisfactory yields of this hydr ophobic and poorly soluble protein. Structural and functional properties of SIN were analyzed and compared with the homologous region of human phospho lamban (PLB) comprising residues Ala(24)-Leu(52) (PLB(24-52)), the regulato ry protein of the cardiac sarcoplasmic reticulum Ca-ATPase. Circular dichro ism spectroscopy showed that SLN is a predominantly alpha -helical protein and that the secondary structure is highly resistant to SDS and thermal den aturation. In this respect SLN is remarkably similar to PLB-(24-52). Howeve r, SLN is monomeric in SDS gels, whereas PLB-(24-52) shows a monomer-pentam er equilibrium typical for native PLB. Analytical ultracentrifugation exper iments revealed that SLN oligomerizes in the presence of the nonionic deter gents octylpolyoxyethylene and octyl glucoside in a concentration-dependent manner. No plateau was observed, and a pentameric state was only reached a t much higher protein concentrations compared with PLB-(24-52). Chemical cr oss-linking showed that also in liposomes SLN has the ability to self-assoc iate to oligomers. PLB-(24-52) specifically oligomerized to pentamers in th e presence of octylpolyoxyethylene as well as in liposomes at low protein c oncentrations. In the presence of octylpolyoxyethylene pentamers were the m ain oligomeric species, whereas in liposomes monomers and dimers were predo minant. Increasing the protein concentration led to self-association of PLB -(24-52) pentamers in the presence of octylpolyoxyethylene. Functional reco nstitution of Ca-ATPase with PLB-(24-52) and SLN in liposomes showed that b oth proteins regulate the Ca-ATPase in a similar manner.