SECONDARY STRUCTURE AND ORIENTATION OF PHOSPHOLAMBAN RECONSTITUTED INSUPPORTED BILAYERS FROM POLARIZED ATTENUATED TOTAL-REFLECTION FTIR SPECTROSCOPY

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 car boxy-terminal transmembrane segment(PLB(26-52)) The relative contents of alpha-helix, beta-strand, and random coil, as well as the spatial o rientations of the alpha-helices of these molecules, reconstituted in dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphat idylcholine (POPC) bilayer membranes, were determined using polarized attenuated total reflection (ATR) Fourier transform infrared (FTIR) sp ectroscopy. The major component of the amide I' bands of PLB and PLB(2 6-52) was centered at 1654-1657 cm(-1) and was assigned to alpha-helix . The fraction of alpha-helix in native PLB was 64-67% (33-35 residues ), and the transmembrane peptide PLB(26-52) contained 73-82% alpha-hel ix (20-22 residues); small fractions of beta- and random structures we re also identified. The orientational order parameter (S) of the alpha -helical component of PLB(26-52) in DMPC was S = 0.86 +/- 0.09, indica ting that the transmembrane helix was oriented approximately perpendic ular to the membrane plane. Assuming the transmembrane domain of PLB r esembles the peptide PLB(26-52), the additional alpha-helical 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 cytoplasm ic helix was tilted from the membrane normal by an angle of 61 +/- 13 degrees or, alternatively, may indicate a wide angular distribution. P LB reconstituted in the supported DMPC bilayers was phosphorylated by the catalytic subunit of protein kinase A, as confirmed by the appeara nce of a new absorbance band at similar to 1200 cm(-1). Phosphorylatio n reduced the alpha-helical content of PLB to 54% (similar to 28 resid ues), though the orientation of the cytoplasmic helix was not signific antly changed. These results, in conjunction with Chou-Fasman secondar y structure prediction, are consistent with a model of PLB composed of a transmembrane helix (residues 33-52), a cytoplasmic helix (most lik ely residues 8-20), and a small intervening beta-sheet between residue s 22 and 32 as well as a random coil at the amino terminus of the prot ein.