Secondary Structure and Orientation of Phospholamban Reconstituted in Supported Bilayers from Polarized Attenuated Total Reflection FTIR Spectroscopy
| Autor: | Laxma G. Reddy, Lukas K. Tamm, David L. Stokes, Suren A. Tatulian, Larry R. Jones |
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| Rok vydání: | 1995 |
| Předmět: |
Models
Molecular Chemistry Bilayer Calcium-Binding Proteins Lipid Bilayers Molecular Sequence Data Biochemistry Protein Structure Secondary Recombinant Proteins Random coil Phospholamban Transmembrane domain Crystallography chemistry.chemical_compound Spectroscopy Fourier Transform Infrared Helix Phosphatidylcholines Amino Acid Sequence Dimyristoylphosphatidylcholine Protein secondary structure Integral membrane protein POPC Mathematics |
| Zdroj: | Biochemistry. 34:4448-4456 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | 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 a-helix, P-strand, and random coil, as well as the spatial orientations of the a-helices of these molecules, reconstituted in dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-Zoleoylphosphatidylcholine (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-' and was assigned to a-helix. The fraction of a-helix in native PLB was 64-67% (33-35 residues), and the transmembrane peptide PLB26-52 contained 73-82% a-helix (20-22 residues); small fractions of P- and random structures were also identified. The orientational order parameter (S) of the a-helical component of PLB26-52 in DMPC was S = 0.86 f 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 a-helical residues in PLB were assigned to the cytoplasmic helix and determined to have an order parameter S = -0.15 f 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-'. Phosphorylation reduced the a-helical 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 P-sheet between residues 22 and 32 as well as a random coil at the amino terminus of the protein. |
| Databáze: | OpenAIRE |
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