Total synthesis and functional properties of the membrane‐intrinsic protein phospholamban

The membrane‐intrinsic protein phospholamban (PLN), the regulatory protein of the sarcoplasmic reticulum (SR) Ca²⁺‐ATPase, was chemically synthesized. The synthesis was accomplished by double couplings and efficient capping procedures, thus eliminating hydrophobic failure sequences. The crude peptide was purified by high‐performance liquid chromatographic ion exchange and gel permeation chromatography in chloroform–methanol mixtures. Ion spray mass spectroscopy showed that the product had the correct molecular mass. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis runs produced the typical monomer–pentamer structural pattern. A predominantly helical CD spectrum was obtained in 0.075% C₁₂E₈ (67.9% helix, 1.8% beta, 12.2% turn, 18.1% random coil). Synthetic PLN was phosphorylated in detergent solutions by protein kinase A with a stoichiometry close to 1:1 (Pi to PLN monomer). Reconstitution of the isolated skeletal muscle SR Ca²⁺ ATPase in phosphatidylcholine membranes in the presence of PLN using the freezing and thawing technique yielded a preparation with lower Ca²⁺‐dependent ATPase activity. The inhibition was mainly due to a decrease in the affinity (K_m(Ca)) of the ATPase for Ca²⁺ and was partially reversed by PLN phosphorylation with protein kinase A. By contrast, addition of PLN to diluted intact SR vesicles uncoupled the Ca²⁺‐transport reaction, suggesting an ionophoric effect of PLN. Because this effect was observed at very high PLN‐to‐SR vesicle ratios and was not influenced by PLN phosphorylation, its biological function is doubtful.