PHOSPHORYLATION-INDUCED STRUCTURAL-CHANGE IN PHOSPHOLAMBAN AND ITS MUTANTS, DETECTED BY INTRINSIC FLUORESCENCE

We have used intrinsic fluorescence to test the hypothesis that phosph orylation induces a conformational change in phospholamban (PLB), a re gulatory protein in cardiac sarcoplasmic reticulum (SR). Phosphorylati on of PLB, which relieves inhibition of the cardiac Ca-ATPase, has bee n shown to decrease the mobility of PLB in sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE). In the present study, we fo und that this mobility shift depends on the acrylamide concentration i n the gel, suggesting that phosphorylation increases the effective Sto kes radius. To further characterize this structural change, we perform ed spectroscopic experiments under the conditions of SDS-PAGE. CD indi cated that phosphorylation at Ser-16 does not change PLB's secondary s tructure significantly. However, the fluorescence of Tyr-6 in the cyto plasmic 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 mut ation (L37A) in the transmembrane domain, which disrupts PLB pentamers and produces monomers in SDS-PAGE and in lipid bilayers, showed simil ar phosphorylation effects on fluorescence, indicating that subunit in teractions within PLB are not crucial for the observed conformational change in SDS. When PLB was reconstituted into dioleoylphosphatidylcho line (DOPC) Lipid bilayers, similar phosphorylation effects in fluores cence were observed, suggesting that PLB behaves similarly in response to phosphorylation in both detergent and lipid environments. We concl ude that phosphorylation induces a structural change within the PLB pr otomer that decreases the solvent accessibility of Tyr-6. The similari ty of this structural change in monomers and pentamers is consistent w ith models in which the PLB monomer is sufficient for the phosphorylat ion-dependent regulation of the Ca-ATPase.