M. Li et al., PHOSPHORYLATION-INDUCED STRUCTURAL-CHANGE IN PHOSPHOLAMBAN AND ITS MUTANTS, DETECTED BY INTRINSIC FLUORESCENCE, Biochemistry, 37(21), 1998, pp. 7869-7877
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.