Secondary Structure and Orientation of Phospholamban Reconstituted in Supported Bilayers from Polarized Attenuated Total Reflection FTIR Spectroscopy

Suren A. Tatulian, Laxma G. Reddy, David L. Stokes, Lukas K. Tamm, Larry R. Jones

Research output: Contribution to journalArticle

106 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)4448-4456
Number of pages9
Issue number13
StatePublished - Sep 1 1995

ASJC Scopus subject areas

  • Biochemistry

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