title: Secondary structure and orientation of phospholamban reconstituted in supported bilayers from polarized attenuated total reflection FTIR spectroscopy.
abstract: 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 alpha-helix, beta-strand, and random coil, as well as the spatial orientations of the alpha-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 alpha-helix. The fraction of alpha-helix in native PLB was 64-67% (33-35 residues), and the transmembrane peptide PLB26-52 contained 73-82% alpha-helix (20-22 residues); small fractions of beta- and random structures were also identified. The orientational order parameter (S) of the alpha-helical 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 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 cytoplasmic helix was tilted from the membrane normal by an angle of 61 +/- 13 degrees or, alternatively, may indicate a wide angular distribution.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Health Sciences Center, Charlottesville 22908, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Tatulian
authors.forename: L R
authors.initials: LR
authors.lastname: Jones
authors.forename: L G
authors.initials: LG
authors.lastname: Reddy
authors.forename: D L
authors.initials: DL
authors.lastname: Stokes
authors.forename: L K
authors.initials: LK
authors.lastname: Tamm
mesh_terms: D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D008433:Mathematics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D000099211:Phospholamban
chemical_list: D002135:Calcium-Binding Proteins; D004134:Dimyristoylphosphatidylcholine; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D000099211:Phospholamban; C028694:1-palmitoyl-2-oleoylphosphatidylcholine