Structural Characterization of Phospholamban in Cardiac Sarcoplasmic Reticulum Membranes by Cross-Linking

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Abstract
The native form of phospholamban in cardiac sarcoplasmic reticulum membranes was investigated using photosensitive heterobifunctional cross-linkers, both cleavable and noncleavable, and common protein modifiers. The photosensitive heterobifunctional cleavable cross-linker ethyl 4-azidophenyl-1,4-dithiobutyrimidate was used in native SR vesicles and it cross-linked phospholamban into an apparent phospholamban–-phospholamban dimer and into an ∼110,000-Da species. The phospholamban dimer migrated at ∼12,000 Da on sodium dodecyl sulfate-polyacrylamide gels, and upon cleavage of the cross-linker before electrophoresis the dimer disappeared. The ∼110,000-Da cross-linked species was not affected by boiling in sodium dodecyl sulfate prior to electrophoresis. This cross-linked form of phospholamban migrated ∼5500 Da above the Ca2+ -ATPase, which was visualized using fluorescein 5′-isothiocyanate, a fluorescent marker that binds specifically to the Ca2+ -ATPase. p-Azidophenacyl bromide, iodoacetic acid, and N-ethylmaleimide, all of which react with sulfhydryl groups, were also employed to further characterize phospholamban in native sarcoplasmic reticulum membranes. Cross-linking with p-azidophenacyl bromide resulted in only monomeric and dimeric forms of phospholamban as observed on sodium dodecyl sulfate-polyacrylamide gels. Iodoacetic acid and N-ethylmaleimide were found to be effective in disrupting the pentameric form of phospholamban only when reacted with sodium dodecyl sulfate solubilized sarcoplasmic reticulum. In view of these findings, the amino acid sequence of phospholamban was examined for possible protein-protein interaction sites. Analysis by hydropathic profiling and secondary structure prediction suggests that the region of amino acids 1–14 may form an amphipathic α helix and the hydrophobic surface on one of its sites could interact with the reciprocal hydrophobic surface of another protein, such as the Ca2+ -ATPase.

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