We used EPR spectroscopy to probe directly the interaction between phospholamban 
(PLB) and its regulatory target, the sarcoplasmic reticulum Ca-ATPase (SERCA). 
Synthetic monomeric PLB was prepared with a single cytoplasmic cysteine at 
residue 11, which was then spin labeled. PLB was reconstituted into membranes in 
the presence or absence of SERCA, and spin label mobility and accessibility were 
measured. The spin label was quite rotationally mobile in the absence of SERCA, 
but became more restricted in the presence of SERCA. SERCA also decreased the 
dependence of spin label mobility on PLB concentration in the membrane, 
indicating that SERCA reduces PLB-PLB interactions. The spin label MTSSL, 
attached to Cys11 on PLB by a disulfide bond, was stable at position 11 in the 
absence of SERCA. In the presence of SERCA, the spin label was released and a 
covalent bond was formed between PLB and SERCA, indicating direct interaction of 
one or more SERCA cysteine residues with Cys11 on PLB. The accessibility of the 
PLB-bound spin label IPSL to paramagnetic agents, localized in different phases 
of the membrane, indicates that SERCA greatly reduces the level of interaction 
of the spin label with the membrane surface. We propose that the cytoplasmic 
domain of PLB associates with the lipid surface, and that association with SERCA 
induces a major conformational change in PLB in which the cytoplasmic domain is 
drawn away from the lipid surface by SERCA.