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 C-13 and H-2 in the transmembrane domain and incorporated into dimyristoylphosphatidylcholine (DMPC) bilayers. Wide line H-2 NMR and C-13 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 H-1-C-13 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 mu s, whereas the rotation of the other species was comparatively slow (tau(R) similar to 60 mu s). 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.