Detection of Sarcolipin Dimerization and SERCA Binding using FRET Microscopy

We have monitored molecular interactions of sarcolipin (SLN) and the sarcoplasmic reticulum Ca-ATPase (SERCA) by measuring Forster resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). SLN regulates contractility in cardiac and skeletal muscle by regulating SERCA calcium uptake and perhaps by acting as an ion channel. Here, live cell FRET microscopy provided novel evidence for self-association of SLN and for regulatory complex formation between SERCA and SLN. Fluorescence photobleaching revealed that SLN self-assembles into dimers but not higher oligomers, and that SERCA and SLN monomers bind together in a 1:1 binary complex. FRET between CFP-SLN:YFP-SLN and CFP-SERCA:YFP-SLN exhibited a hyperbolic dependence on protein concentration, with maximum efficiency of 62 ± 1% and 52 ± 1%, respectively. The concentration of half-maximal FRET was 7.6 ± 0.4 AU for SLN:SLN and 17.0 ± 1.0 AU for SERCA:SLN, demonstrating that the binding affinity of SLN:SLN homo-dimers is 2.2-fold greater than SERCA:SLN hetero-dimers. Mutating SLN residue Ile-17 to Ala (I17A) decreased maximum FRET for SERCA:SLN by 14 ± 4% with no change in binding affinity, indicating that the I17A-SLN mutation causes a structural rearrangement with distance increase of 5 ± 1 A within the binary regulatory complex. Addition of wild-type SERCA (cardiac and fast-twitch isoforms) decreased the binding affinity of the SLN:SLN interaction by 24 ± 6% with no change in maximum FRET, indicating that SLN monomers are in competition between dimerization and SERCA binding. We propose that SLN exists as monomers, homo-dimers, and hetero-dimers in sarcoplasmic reticulum membranes, and that Ile-17 of SLN acts as an allosteric switch in the regulatory complex with SERCA.