Probing the Molecular Mechanism of SERCA-PLB Regulation by Time-Resolved FRET

Abstract

We are using solid-phase peptide synthesis, membrane reconstitution, an enzyme-coupled Ca-ATPase activity assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA.