Secondary structure of detergent-solubilized phospholamban, a phosphorylatable, oligomeric protein of cardiac sarcoplasmic reticulum

Heather K B Simmerman, D. Eugene Lovelace, Larry Jones

Research output: Contribution to journalArticle

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Abstract

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 α-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 α-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.

Original languageEnglish
Pages (from-to)322-329
Number of pages8
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume997
Issue number3
DOIs
StatePublished - Aug 31 1989

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Sarcoplasmic Reticulum
Detergents
Proteins
Circular Dichroism
Sodium Dodecyl Sulfate
Circular dichroism spectroscopy
Phosphorylation
Spectrum Analysis
phospholamban
Aromatic Amino Acids
Denaturation
Glycols
Dichroism
Cyclic AMP-Dependent Protein Kinases
Phenylalanine
Boiling liquids
Serine
Tyrosine
Absorption spectra
Catalytic Domain

Keywords

  • Circular dichroism
  • Phospholamban
  • Protein phosphorylation

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Structural Biology

Cite this

Secondary structure of detergent-solubilized phospholamban, a phosphorylatable, oligomeric protein of cardiac sarcoplasmic reticulum. / Simmerman, Heather K B; Eugene Lovelace, D.; Jones, Larry.

In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, Vol. 997, No. 3, 31.08.1989, p. 322-329.

Research output: Contribution to journalArticle

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N2 - 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 α-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 α-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.

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