Mutation and phosphorylation change the oligomeric structure of phospholamban in lipid bilayers

Rǎzvan L. Cornea, Larry Jones, Joseph M. Autry, David D. Thomas

Research output: Contribution to journalArticle

153 Citations (Scopus)

Abstract

Phospholamban (PLB), a 52-residue protein integral to the cardiac sarcoplasmic reticulum, is a key regulator of the Ca pump. PLB has been shown to form pentamers in the denaturing detergent sodium dodecyl sulfate (SDS), but its oligomeric state in the natural environment of the lipid membrane remains unknown. In order to address this issue, we performed electron paramagnetic resonance (EPR) experiments on two types of lipid-reconstituted, recombinant PLB: wild type (WT PLB) and a mutant substituted with alanine at leucine 37 (L37A PLB), whose propensity to oligomerize in SDS is greatly diminished. The lipid used in reconstitution was dioleoylphosphatidylcholine (DOPC) doped with a phospholipid spin-label that detects protein contact. EPR spectroscopy was used to determine the fraction of the total lipid molecules in contact with PLB. Our results show that, in phospholipid bilayers, WT PLB is oligomeric (effective oligomeric size of 3.52 ± 0.71), while L37A PLB is monomeric (effective oligomeric size of 1.15 ± 0.15). Thus, the oligomeric states of these proteins in the lipid membrane are remarkably similar to those in SDS solution. In particular, the point mutation in L37A PLB greatly destabilizes the PLB oligomer. Phosphorylation of PLB by protein kinase A, which has been shown to relieve inhibition of the cardiac Ca pump, changes the lipid-PLB interactions, decreasing the number of lipids restricted by contact with protein. The results are consistent with a phosphorylation- dependent increase of the effective oligomer size of WT PLB from 3.52 to 5.34 and of L37A PLB from 1.15 to 1.91. These phosphorylation effects were abolished in a medium with a high ionic strength. We conclude that the oligomeric states of PLB in lipid membranes are in a dynamic equilibrium that is perturbed by phosphorylation due to reduced electrostatic repulsion among PLB protomers.

Original languageEnglish
Pages (from-to)2960-2967
Number of pages8
JournalBiochemistry
Volume36
Issue number10
DOIs
StatePublished - Mar 11 1997

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Phosphorylation
Lipid bilayers
Lipid Bilayers
Mutation
Lipids
Membrane Lipids
Sodium Dodecyl Sulfate
Electron Spin Resonance Spectroscopy
phospholamban
Oligomers
Phospholipids
Paramagnetic resonance
Proteins
Pumps
Spin Labels
Protein Subunits
Sarcoplasmic Reticulum
Cyclic AMP-Dependent Protein Kinases
Static Electricity
Point Mutation

ASJC Scopus subject areas

  • Biochemistry

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Mutation and phosphorylation change the oligomeric structure of phospholamban in lipid bilayers. / Cornea, Rǎzvan L.; Jones, Larry; Autry, Joseph M.; Thomas, David D.

In: Biochemistry, Vol. 36, No. 10, 11.03.1997, p. 2960-2967.

Research output: Contribution to journalArticle

Cornea, Rǎzvan L. ; Jones, Larry ; Autry, Joseph M. ; Thomas, David D. / Mutation and phosphorylation change the oligomeric structure of phospholamban in lipid bilayers. In: Biochemistry. 1997 ; Vol. 36, No. 10. pp. 2960-2967.
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abstract = "Phospholamban (PLB), a 52-residue protein integral to the cardiac sarcoplasmic reticulum, is a key regulator of the Ca pump. PLB has been shown to form pentamers in the denaturing detergent sodium dodecyl sulfate (SDS), but its oligomeric state in the natural environment of the lipid membrane remains unknown. In order to address this issue, we performed electron paramagnetic resonance (EPR) experiments on two types of lipid-reconstituted, recombinant PLB: wild type (WT PLB) and a mutant substituted with alanine at leucine 37 (L37A PLB), whose propensity to oligomerize in SDS is greatly diminished. The lipid used in reconstitution was dioleoylphosphatidylcholine (DOPC) doped with a phospholipid spin-label that detects protein contact. EPR spectroscopy was used to determine the fraction of the total lipid molecules in contact with PLB. Our results show that, in phospholipid bilayers, WT PLB is oligomeric (effective oligomeric size of 3.52 ± 0.71), while L37A PLB is monomeric (effective oligomeric size of 1.15 ± 0.15). Thus, the oligomeric states of these proteins in the lipid membrane are remarkably similar to those in SDS solution. In particular, the point mutation in L37A PLB greatly destabilizes the PLB oligomer. Phosphorylation of PLB by protein kinase A, which has been shown to relieve inhibition of the cardiac Ca pump, changes the lipid-PLB interactions, decreasing the number of lipids restricted by contact with protein. The results are consistent with a phosphorylation- dependent increase of the effective oligomer size of WT PLB from 3.52 to 5.34 and of L37A PLB from 1.15 to 1.91. These phosphorylation effects were abolished in a medium with a high ionic strength. We conclude that the oligomeric states of PLB in lipid membranes are in a dynamic equilibrium that is perturbed by phosphorylation due to reduced electrostatic repulsion among PLB protomers.",
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