Secondary structure of detergent-solubilized phospholamban, a phosphorylatable, oligomeric protein of cardiac sarcoplasmic reticulum.
The structure of phospholamban, a 30-kDa oligomeric protein integral to cardiac sarcoplasmic reticulum, was probed using ultraviolet absorbance and circular dichroism spectroscopy. Purified phospholamban was examined in three detergents: octyl glucoside, n-dodecyloctaethylene glycol monoether (C12E8) and sodium dodecyl sulfate (SDS). Ultraviolet absorption spectra of phospholamban reflected its aromatic amino acid content: absorption peaks at 275-277 nm and 253, 259, 265 and 268 nm were attributed to phospholamban's one tyrosine and two phenylalanines, respectively. Phospholamban phosphorylated at serine 16 by the catalytic subunit of cAMP-dependent protein kinase exhibited no absorbance changes when examined in C12E8 or SDS. Circular dichroism spectroscopy at 250-190 nm demonstrated that phospholamban possesses a very high content of alpha-helix in all three detergents and is unusually resistant to denaturation. Dissociation of phospholamban subunits by boiling in SDS increased the helical content, suggesting that the highly ordered structure is not dependent upon oligomeric interactions. The purified COOH-terminal tryptic fragment of phospholamban, containing residues 26-52 and comprising the hydrophobic, putative membrane-spanning domain, also exhibited a circular dichroism spectrum characteristic of alpha-helix. Circular dichroism spectra of phosphorylated and dephosphorylated phospholamban were very similar, indicating that phosphorylation does not alter phospholamban secondary structure significantly. The results are consistent with a two-domain model of phospholamban in which each domain contains a helix and phosphorylation may act to rotate one domain relative to the other.
Simmerman
H K
HK
Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis 46202.
Lovelace
D E
DE
Jones
L R
LR
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D003902:Detergents; D004285:Dogs; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D000099211:Phospholamban
D000251:Adenosine Triphosphatases; D002135:Calcium-Binding Proteins; D003902:Detergents; D046911:Macromolecular Substances; D011494:Protein Kinases; D000099211:Phospholamban