Structural Dynamics of SERCA and Phospholamban by TR-FRET and TIRF

Abstract

We used fluorescence to investigate the structure and dynamics of phospholamban (PLB) and its inhibited target, sarcoplasmic reticulum Ca-ATPase (SERCA). Time-resolved fluorescence energy transfer (TR-FRET) was used to study the oligomeric interaction between PLB and SERCA. Previous work on our lab has shown that PLB is primarily pentameric but SERCA binds preferentially to the monomeric form in lipid vesicles. Recent EM studies suggest that the PLB pentamer might also bind to SERCA. We tested this hypothesis by labeling SERCA at C674 with a fluorescent donor (TMRIA) and labeling PLB at K3 with a non-fluorescent acceptor (MGITC), then reconstituting the proteins into lipid vesicles and performing TR-FRET as a function of the fraction of acceptor-labeled PLB (xA), keeping the total PLB/SERCA molar ratio constant at 10. Simulations showed that if a PLB monomer binds to SERCA, the dependence of FRET on xA should be linear, but the binding of a PLB oligomer should produce distinct curvature in the plot. The observed plot was quite linear, and was indistinguishable from control experiments using a monomeric mutant of PLB. We conclude that PLB binds to SERCA only as a monomer. We used polarized TIRF to study the structure of PLB in the presence and absence of SERCA in a supported lipid bilayer. Polarized TIRF data shows that the cytoplasmic helix of PLB, labeled with bifunctional rhodamine, is close to be parallel to the membrane surface. SERCA binding lifts the cytoplasmic helix away from the membrane surface. These results have important implications for the design of PLB mutants to be used in gene therapy for heart failure.