We are using site-directed spin-labeling (SDSL) and EPR spectroscopy to study the structural dynamics of phospholamban (PLB), a 52-residue integral membrane protein that regulates the SR calcium ATPase (SERCA). PLB binds and inhibits SERCA at sub-micromolar calcium concentrations, while phosphorylation of PLB at Ser16 relieves this inhibition without dissociating the two proteins (Mueller et al., 2004). Employing solid-state peptide synthesis, we have created PLB analogs in which the spin-labeled amino acid TOAC is substituted for residues along the backbone. Doubly-labeled proteins were studied by DEER, a pulsed EPR experiment that can measure inter-spin-label distances from 2 to 7 nm. Our results agree with previously published EPR dynamics data showing that PLB exists in both a compact, ordered (T) state and an extended, dynamically disordered (R) state (Karim et al., 2006). Alone, PLB primarily occupies the (T) state, while this equilibrium shifts in favor of the (R) state upon SERCA binding or PLB phosphorylation. However, SERCA-bound PLB becomes more ordered and compact upon phosphorylation. We are also using relaxation enhancement to study the movement of PLB's single transmembrane (TM) helix relative to the membrane plane. In these experiments, the spin-lattice relaxation rate of excited spins is enhanced by the presence of paramagnetic relaxation agents (PRAs), which collide with these spins and cause them to relax faster. For spin-labels incorporated into the TM domain, PLB motions that reposition this helix will make the spin-label more or less accessible to water-soluble PRAs, while having the opposite effect for lipid-soluble PRAs. The magnitude of change in the relaxation rate can be used to gauge the movement of the TM helix upon SERCA binding and following phosphorylation. With these experiments, we are constructing a more complete model of PLB dynamics during its interaction with SERCA.