Muscle contraction and relaxation is regulated by calcium flux between the sarcoplasmic reticulum and the cytoplasm. Subsequent to muscle contraction, calcium must be sequestered to the sarcoplasmic reticulum in order for muscle relaxation to occur. The sarco-endoplasmic reticulum Ca-ATPase (SERCA) is a P-type ATPase embedded in the SR membrane which uses ATP hydrolysis to pump calcium back into the SR lumen to facilitate muscle relaxation. In cardiac muscle, SERCA activity is regulated by phospholamban (PLB) a 52-residue integral membrane protein which exists in a dynamic equilibrium between monomeric and pentameric species. Previous data have shown that monomeric PLB is the primary regulator of SERCA activity but recent publications have proposed that the PLB pentamer may also bind to and inhibit SERCA activity. This inhibition can be relieved by phosphorylation of PLB at Ser16, although the mechanism is not known.
Electron Paramagnetic Resonance (EPR) experiments were designed to test two proposed models of the PLB pentamer, the pinwheel and bellflower. Dynamics data using the TOAC amino acid spin label showed that, like the monomer, the pentamer is in a dynamic equilibrium between ordered (T) and dynamically disordered (R) states, with the T state being predominant. Accessibility of spin labels attached to the cytoplasmic domain to the lipid bilayer showed that, like the monomer, the pentamer cytoplasmic domains strongly interact with the lipid bilayer surface. Finally, pulsed EPR (DEER) experiments measuring long range distances between spin labels attached to the cytoplasmic domain showed a bimodal distance distribution with centers at 3 and 5 nm. All of these data support the pinwheel model.
To investigate SERCA binding and phosphorylation affects on PLB dynamics, a monomeric mutant, AFA-PLB, was spin labeled with TOAC either the 11 position in the cytoplasmic domain or 36 in the transmembrane domain. Conventional EPR measurements showed that phosphorylation induced and order-to-disorder conformational change in the cytoplasmic domain and that SERCA preferentially binds the PLB R state. Phosphorylation of SERCA bound PLB resulted in a disorder-to-order conformational change, suggesting that pPLB is still bound to SERCA. Conventional dynamics from 36-TOAC in the transmembrane indicated a stable helix which was unaffected by phosphorylation or SERCA binding. Dipolar EPR measurements revealed that phosphorylation of PLB in the absence of SERCA induces oligomerization and that SERCA destabilizes the pPLB oligomer. Saturation Transfer EPR data which measures the rotational diffusion of PLB in the lipid bilayer supported the conclusion that phosphorylation of PLB in the absence of SERCA induces oligomerization and showed directly that phosphorylated PLB is still bound to active SERCA. These data support the model that phosphorylation dependent relief of SERCA inhibition does not require dissociation of the SERCA-PLB complex, but is rather the result of a structural change in the complex.
University of Minnesota Ph.D. dissertation. July 2010. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: David D. Thomas. 1 computer file (PDF); xi, 134 pages. Ill. (some col.).
Torgersen, Kurt Daniel.
Cardiac calcium transport regulation probed by electron paramagnetic resonance spectroscopy..
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