Sarcoplasmic reticulum Ca2+ATPase (SERCA) transports Ca2+ ions from the cytosol of cardiomyocytes into the SR, making it a crucial regulator of cardiac muscle function. SERCA is inhibited by phospholamban (PLN), a membrane-spanning protein consisting of a transmembrane (TM) helix connected to an amphipathic helix by a short loop. Phosphorylation of PLN at Ser16 relieves the inhibition of SERCA but does not dissociate the PLN-SERCA complex. Owing to their pivotal role in cardiac muscle function, SERCA and PLN have received much attention as therapeutic targets. In one such study, a pseudo-phosphorylated S16E mutant of PLN was successfully used to relieve the effects of heart failure in hamsters (Nat. Med., 2002, 8, 864-871). Ongoing efforts in our lab are aimed to design new PLN mutants that bind tightly with SERCA, but relieve inhibition (i.e., mimic Ser16 phosphorylation).A recent study refined the structure and topology of PLN in a lipid bilayer environment using a hybrid solution and solid state NMR approach (PNAS, 2009, 106, 25, 10165-101670). The tilt angle of the PLN TM helix in DOPC/DOPE bilayers was determined to be 24o with respect to the bilayer normal. In this study, we reconstituted PLN into magnetically aligned bicelles and used PISEMA experiments to show that the topology of PLN is strongly dependent on hydrophobic mismatch, since mutations in the PLN sequence and changes in the bilayer composition alter the TM helix tilt angle. Taken with results from our lab that correlated PLN structural dynamics to inhibitory function on SERCA (J. Biol. Chem., 2007, 282, 51, 37205-14), the differences in topology presented in this work introduce a new dimension in the study of PLN mutants that could be crucial for understanding the complex structural features of PLN that are important for SERCA regulation.