Chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) have been used to analyze the structure and function of phospholamban (PLB), a 52-residue integral membrane protein that regulates the calcium pump (Ca-ATPase) in cardiac sarcoplasmic reticulum (SR). PLB exists in equilibrium between monomeric and pentameric forms, as observed by SDS-PAGE, EPR, and fluorescence. It has been proposed that inhibition of the pump is due primarily to the monomeric form, with both pentameric stability and inhibition dependent primarily on the transmembrane (TM) domain. To test these hypotheses, we have studied the physical and functional properties of a synthetic null-cysteine PLB analogue that is entirely monomeric on SDS-PAGE, and compared it with the synthetic null-cysteine TM domain (residues 26-52). The TM domain was found to be primarily oligomeric on SDS-PAGE, and boundary lipid spin label analysis in lipid bilayers verified that the isolated TM domain is more oligomeric than the full-length parent molecule. These results indicate that the stability of the PLB pentamer is due primarily to attractive interactions between hydrophobic TM domains, overcoming the repulsive electrostatic interactions between the cationic cytoplasmic domains (residues 1-25). When reconstituted into liposomes containing the Ca-ATPase, the null-cysteine TM domain had the same inhibitory function as that of the full-length parent molecule. We conclude that the TM domain of PLB is sufficient for inhibitory function, the oligomeric stability of PLB does not determine its inhibitory activity, and the three Cys residues in the TM domain are not required for inhibitory function.

To test models for the pentameric structure of phospholamban (PLB) and study its structure and molecular dynamics in SDS solution, we characterized recombinant PLB and several of its mutants by (a) reactivity of cysteine residues toward DTNB [5, 5'-dithiobis(2-nitrobenzoic acid)] and a thiol-reactive spin label, (b) oligomeric state on SDS-PAGE, and (c) EPR of the spin-labeled proteins. WT-PLB has three cysteine residues (36, 41, and 46), all located in the hydrophobic C-terminal transmembrane region. In SDS at pH 7.5, exhaustive reaction with either sulfhydryl reagent resulted in essentially 2 mol of cysteine reacted/mol of WT-PLB, with only slight destabilization of the native pentameric structure. When WT-PLB was denatured in guanidine at pH 8.1, all three cysteines reacted, disrupting the pentamer, which was restored upon cleavage of the disulfide bonds with DTT. In the tetrameric mutant C41L-PLB, the two remaining cysteine residues reacted, reversibly destabilizing the tetramer. In the monomeric mutant L37A-PLB, all three cysteines reacted. The pentameric double cysteine replacement mutant C36,46A-PLB showed negligible reactivity. We conclude that Cys-41 is the unreactive cysteine in PLB and is located at a crucial site for the maintenance of the pentameric structure. EPR spectra in SDS of spin-labeled WT-PLB and mutants correlate with the oligomeric state on SDS-PAGE; oligomeric proteins show decreased spin-label mobility compared with monomers. Molecular dynamics calculations were used to construct an atomic model for the transmembrane region of the PLB pentamer, constrained by previous mutagenesis results and the results of the present study. We conclude that (a) the mobilities of spin-labels attached to PLB and its mutants are sensitive to oligomeric state and (b) the pattern of cysteine reactivity, spin-label mobility, and oligomeric state supports a structural model for the PLB pentamer in which interactions between each pair of subunits are stabilized by a leucine-isoleucine zipper.

In order to test molecular models of cardiac calcium transport regulation, we have used spectroscopy to probe the structures, dynamics, and interactions of the Ca pump (Ca-ATPase) and phospholamban (PLB) in cardiac sarcoplasmic reticulum (SR) and in reconstituted membranes. Electron paramagnetic resonance (EPR) and phosphorescence of probes bound to the Ca pump show that the activity of the pump is quite sensitive to its oligomeric interactions. In cardiac SR, PLB aggregates and inhibits the pump, and both effects are reversed by PLB phosphorylation. Previous analyses of PLB's oligomeric state were only in detergent solutions, so we used EPR and fluorescence to determine the oligomeric structure of PLB in its native state in lipid bilayers. Wild-type PLB is primarily oligomeric in the membrane, while the mutant L37A-PLB is monomeric. For both proteins, phosphorylation shifts the dynamic monomer-oligomer equilibrium toward oligomers, and induces a similar structural change, as indicated by tyrosine fluorescence; yet L37A-PLB is more effective than wild-type PLB in inhibiting and aggregating the pump. Fluorescence energy transfer shows that the Ca pump increases the fraction of monomeric PLB, indicating that the pump preferentially binds monomeric PLB. These results support a reciprocal aggregation model for Ca pump regulation, in which the Ca pump is aggregated and inhibited by association with PLB monomers, and phosphorylation of PLB reverses these effects while decreasing the concentration of PLB monomers. To investigate the structure of the PLB pentamer in more detail, we measured the reactivities of cysteine residues in the transmembrane domain of PLB, and recorded EPR spectra of spin labels attached to these sites. These results support an atomic structural model, based on molecular dynamics simulations and mutagenesis studies, in which the PLB pentamer is stabilized by a leucine-isoleucine zipper within the transmembrane domain.

It has been postulated that the hydrophobic loop of actin (residues 262-274) swings out and inserts into the opposite strand in the filament, stabilizing the filament structure. Here, we analyzed the hydrophobic loop dynamics utilizing four mutants that have cysteine residues introduced at a single location along the yeast actin loop. Lateral, copper-catalyzed disulfide cross-linking of the mutant cysteine residues to the native C374 in the neighboring strand within the filament was fastest for S265C, followed by V266C, L267C, and then L269C. Site-directed spin labeling (SDSL) studies revealed that C265 lies closest to C374 within the filament, followed by C266, C267, and then C269. These results are not predicted by the Holmes extended loop model of F-actin. Furthermore, we find that disulfide cross-linking destroys L267C and L269C filaments; only small filaments are observed via electron microscopy. Conversely, phalloidin protects the L267C and L269C filaments and inhibits their disulfide cross-linking. Combined, our data indicate that, in solution, the loop resides predominantly in a "parked" position within the filament but is able to dynamically populate other conformational states which stabilize or destabilize the filament. Such states may be exploited within a cell by filament-stabilizing and -destabilizing factors.