Phospholamban (PLN) is the endogenous inhibitor of the sarco(endo)plasmic 
reticulum Ca(2+)-ATPase (SERCA), the integral membrane enzyme responsible for 
70 % of the removal of Ca(2+) from the cytosol, inducing cardiac muscle 
relaxation in humans. Dysfunctions in SERCA:PLN interactions have been 
implicated as having a critical role in cardiac disease, and targeting Ca(2+) 
transport has been demonstrated to be a promising avenue in treating conditions 
of heart failure. Here, we designed a series of new mutants able to tune SERCA 
function, targeting the loop sequence that connects the transmembrane and 
cytoplasmic helices of PLN. We found that a variable degree of loss of 
inhibition mutants is attainable by engineering glycine mutations along PLN's 
loop domain. Remarkably, a double glycine mutation results in a complete 
loss-of-function mutant, fully mimicking the phosphorylated state of PLN. Using 
nuclear magnetic resonance spectroscopy, we rationalized the effects of these 
mutations in terms of entropic control on PLN function, whose inhibitory 
function can be modulated by increasing its conformational dynamics. However, if 
PLN mutations go past a threshold set by the phosphorylated state, they break 
the structural coupling between the transmembrane and cytoplasmic domains, 
resulting in a species that behaves as the inhibitory transmembrane domain 
alone. These studies provide new potential candidates for gene therapy to 
reverse the effects of heart failure.