We have detected directly the interactions of sarcolipin (SLN) and the sarcoplasmic reticulum Ca-ATPase (SERCA) by measuring fluorescence resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (donor) and yellow fluorescent protein (acceptor). SLN is a membrane protein that helps control contractility by regulating SERCA activity in fast-twitch and atrial muscle. Here we used FRET microscopy and spectroscopy with baculovirus expression in insect cells to provide direct evidence for: 1) oligomerization of SLN and 2) regulatory complex formation between SLN and the fast-twitch muscle Ca-ATPase (SERCA1a isoform). FRET experiments demonstrated that SLN monomers self-associate into dimers and higher order oligomers in the absence of SERCA, and that SLN monomers also bind to SERCA monomers in a 1:1 binary complex when the two proteins are coexpressed. FRET experiments further demonstrated that the binding affinity of SLN for itself is similar to that for SERCA. Mutating SLN residue isoleucine-17 to alanine (I17A) decreased the binding affinity of SLN self-association and converted higher order oligomers into monomers and dimers. The I17A mutation also decreased SLN binding affinity for SERCA but maintained 1:1 stoichiometry in the regulatory complex. Thus, isoleucine-17 plays dual roles in determining the distribution of SLN homooligomers and stabilizing the formation of SERCA-SLN heterodimers. FRET results for SLN self-association were supported by the effects of SLN expression in bacterial cells. We propose that SLN exists as multiple molecular species in muscle, including SERCA-free (monomer, dimer, oligomer) and SERCA-bound (heterodimer), with transmembrane zipper residues of SLN serving to stabilize oligomeric interactions. Sarcolipin (SLN)4 is a 3-kDa membrane protein that reversibly inhibits the activity of the calcium-transporting ATPase (SERCA) in sarcoplasmic reticulum (SR) of fast-twitch, slowtwitch, and atrial muscle (1, 2). SERCA is a 110-kDa membrane protein that relaxes muscle by pumping calcium out of the cytoplasm using energy derived from ATP hydrolysis (3). Transgenic mouse studies have demonstrated that knock-out of SLN enhances atrial contractility (4), cross-expression of SLN inhibits ventricular contractility (2, 5, 6), and ␤-adrenergic stimulation relieves SLN inhibition of contractility (2, 6, 7). Phosphorylation/dephosphorylation of SLN is responsible for SERCA regulation, controlling the rate and amount of calcium loading in SR, which in turn determines the rate of muscle relaxation and the force of subsequent contraction (2, 4 -7). SLN gene expression is up-regulated 2-15-fold in patients with skeletal muscle dysferlinopathy and Takotsubo cardiomyopathy, but down-regulated 2-3-fold in patients with heart failure, atrial fibrillation, and congenital heart defects, indicating that SLN acts as a causative or compensatory factor in human diseases of skeletal and cardiac muscle (8 -12).SLN comprises a single transmembrane (TM) helix, plus a small cytoplasm...

Background: FITC is a useful but underutilized covalent probe of the Ca-ATPase nucleotide-binding site. Results: We measured time-resolved emission, anisotropy, and quenching of FITC-labeled Ca-ATPase. We used enzyme reverse mode to synthesize FITC monophosphate as a tethered, fluorescent ATP analog. Conclusion:The Ca-ATPase active site exhibits increased dynamics when enclosed with bound ATP. Significance: Internal entropy contributes to long range coupling and catalysis in the Ca-ATPase.

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We have used conventional and saturation transfer electron paramagnetic resonance (EPR) to probe the structural dynamics of the integral membrane protein phospholamban (PLB), as a function of phosphorylation and the presence of its regulatory target, the sarcoplasmic reticulum calcium ATPase (SERCA). Our goal is to elucidate the mechanism of SERCA inhibition by PLB, which is needed for the rational design of therapies to improve calcium transport in muscle cells. We used the monomeric mutant AFA-PLB with the rigid electron spin label TOAC incorporated at position 36 in the transmembrane domain, and reconstituted the protein in lipid vesicles. EPR experiments were performed to determine the nanosecond (conventional EPR) and microsecond (saturation transfer EPR) rotational motions of PLB, as modulated by phosphorylation of PLB at serine 16 (pPLB), and/or addition of excess SERCA. Neither SERCA binding nor PLB phosphorylation caused significant changes in conventional EPR spectra of PLB, indicating no effect on the nanosecond flexibility of PLB's transmembrane domain. However, saturation transfer EPR found that addition of SERCA caused a large decrease in microsecond rotational motion for both PLB and pPLB. Phosphorylation of PLB caused no effect in the absence of SERCA and only slight mobilization in the presence of SERCA. These results indicate that both PLB and pPLB bind to SERCA, and PLB phosphorylation only slightly increases the flexibility and/or the dissociation constant of