We have studied the secondary structure of native phospholamban (PLB), a 52-residue integral membrane protein that regulates calcium uptake into the cardiac sarcoplasmic reticulum, as well as its 27-residue carboxy-terminal transmembrane segment (PLB26-52). The relative contents of α-helix, β-strand, and random coil, as well as the spatial orientations of the α-helices of these molecules, reconstituted in dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer membranes, were determined using polarized attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. The major component of the amide I′ bands of PLB and PLB26-52 was centered at 1654-1657 cm-1 and was assigned to αhelix. The fraction of αhelix in native PLB was 64-67% (33-35 residues), and the transmembrane peptide PLB26-52 contained 73-82% αhelix (20-22 residues); small fractions of β- and random structures were also identified. The orientational order parameter (5) of the αhelical component of PLB26-52 in DMPC was S = 0.86 ± 0.09, indicating that the transmembrane helix was oriented approximately perpendicular to the membrane plane. Assuming the transmembrane domain of PLB resembles the peptide PLB26-52, the additional αhelical residues in PLB were assigned to the cytoplasmic helix and determined to have an order parameter S = -0.15 ± 0.30. This may imply that the cytoplasmic helix was tilted from the membrane normal by an angle of 61 ± 13° or, alternatively, may indicate a wide angular distribution. PLB reconstituted in the supported DMPC bilayers was phosphorylated by the catalytic subunit of protein kinase A, as confirmed by the appearance of a new absorbance band at ~1200 cm-1. Phosphorylation reduced the αhelical content of PLB to 54% (~28 residues), though the orientation of the cytoplasmic helix was not significantly changed. These results, in conjunction with Chou-Fasman secondary structure prediction, are consistent with a model of PLB composed of a transmembrane helix (residues 33-52), a cytoplasmic helix (most likely residues 8-20), and a small intervening β-sheet between residues 22 and 32 as well as a random coil at the amino terminus of the protein.
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