We have used intrinsic fluorescence to test the hypothesis that phosphorylation induces a conformational change in phospholamban (PLB), a regulatory protein in cardiac sarcoplasmic reticulum (SR). Phosphorylation of PLB, which relieves inhibition of the cardiac Ca-ATPase, has been shown to decrease the mobility of PLB in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the present study, we found that this mobility shift depends on the acrylamide concentration in the gel, suggesting that phosphorylation increases the effective Stokes radius. To further characterize this structural change, we performed spectroscopic experiments under the conditions of SDS-PAGE. CD indicated that phosphorylation at Set- 16 does not change PLB's secondary structure significantly. However, the fluorescence of Tyr-6 in the cytoplasmic domain of PLB changed significantly upon PLB phosphorylation: phosphorylation increased the fluorescence quantum yield and decreased the quenching efficiency by acrylamide, suggesting a local structural change that decreases the solvent accessibility of Tyr-6. A point mutation (L37A) in the transmembrane domain, which disrupts PLB pentamers and produces monomers in SDS-PAGE and in lipid bilayers, showed similar phosphorylation effects on fluorescence, indicating that subunit interactions within PLB are not crucial for the observed conformational change in SDS. When PLB was reconstituted into dioleoylphosphatidylcholine (DOPC) lipid bilayers, similar phosphorylation effects in fluorescence were observed, suggesting that PLB behaves similarly in response to phosphorylation in both detergent and lipid environments. We conclude that phosphorylation induces a structural change within the PLB protomer that decreases the solvent accessibility of Tyr-6. The similarity of this structural change in monomers and pentamers is consistent with models in which the PLB monomer is sufficient for the phosphorylation-dependent regulation of the Ca-ATPase.
|Original language||English (US)|
|Number of pages||9|
|State||Published - May 26 1998|