Probing the oligomeric state of phospholamban variants in phospholipid bilayers from solid-state NMR measurements of rotational diffusion rates.
Phospholamban (PLB) is a small transmembrane protein that regulates calcium transport across the sarcoplasmic reticulum (SR) of cardiac cells. PLB self-associates into pentamers within sodium dodecyl sulfate (SDS) micelles, but the oligomeric status of PLB in SR membranes is not known. This work has shown that a mutant of PLB, with all native cysteine residues replaced by alanine (Ala-PLB), runs as a monomer on SDS-PAGE gels, in agreement with previous studies [Karim et al. (2000) Biochemistry 39, 10892-10897]. By contrast, a peptide representing the transmembrane domain of the cysteine-free mutant (TM-Ala-PLB) coexists as pentamers, dimers, and monomers on gels. Solid-state NMR methods were used to examine the size and heterogeneity of Ala-PLB and TM-Ala-PLB labeled with (13)C and (2)H in the transmembrane domain and incorporated into dimyristoylphosphatidylcholine (DMPC) bilayers. Wide line (2)H NMR and (13)C cross-polarization magic-angle spinning (CP-MAS) NMR spectra of Ala-PLB and TM-Ala-PLB revealed two distinct species of each of the proteins in the membranes. In the case of Ala-PLB one species was present initially and a second species emerged after 12 h. Measurements of (1)H-(13)C dipolar couplings for the two species of Ala-PLB showed that the rotational diffusion of one species was relatively rapid, defined by a correlation time (tau(R)) of less than 10 micros, whereas the rotation of the other species was comparatively slow (tau(R) approximately 60 micros). These results suggest that although Ala-PLB runs as a monomer on gels, a mixture of different oligomeric forms of the protein, possibly monomers and pentamers, is present in DMPC bilayers. Caution must therefore be exercised in using SDS-PAGE to draw conclusions about the oligomeric state of PLB variants in lipid bilayers.
Hughes
Eleri
E
Faculty of Life Sciences, University of Manchester, P.O. Box 88, Sackville Street, Manchester M60 1QD, UK.
Clayton
Jonathan C
JC
Middleton
David A
DA
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D002247:Carbon Isotopes / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D041961:Deuterium Exchange Measurement; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D008051:Lipid Bilayers / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000737:chemistry*; D010759:Phosphorus Isotopes / Q000378:metabolism; D013816:Thermodynamics; D000099211:Phospholamban
D000409:Alanine; D002135:Calcium-Binding Proteins; D002247:Carbon Isotopes; D003545:Cysteine; D004134:Dimyristoylphosphatidylcholine; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010743:Phospholipids; D010759:Phosphorus Isotopes; D000099211:Phospholamban