Title: Multifrequency EPR Detects Orientation of Calcium Transport Proteins in Lipid Bicelles
Abstract: We have used electron paramagnetic resonance (EPR) at X and Q band to probe the structural dynamics of the integral membrane protein phospholamban (PLB), as a function of phosphorylation and addition of its regulatory target, the sarcoplasmic reticulum calcium ATPase (SERCA). We found previously that PLB remains bound to SERCA after phosphorylation, suggesting that a structural transition within the SERCA-PLB complex is responsible for relief of inhibition. Our current goal is to elucidate this mechanism through orientation and accessibility EPR, supporting rational design of therapies to improve calcium transport in muscle cells. We used the monomeric mutant AFA-PLB with the rigid spin label TOAC incorporated within the transmembrane domain. This protein was reconstituted with purified SERCA into lipid bicelles and magnetically aligned at both X and Q band field strength, allowing for a robust global fitting approach. We measured changes in PLB tilt upon phosphorylation, in the absence and presence of SERCA. Experiments were performed in the Biophysical Spectroscopy Center at the University of Minnesota. This work was funded by grants from NIH (R01 GM27906, P30 AR0507220, and T32 AR007612).