SERCA-phospholamban structural dynamics studied by electron paramagnetic resonance
2013-07
Loading...
View/Download File
Persistent link to this item
Statistics
View StatisticsJournal Title
Journal ISSN
Volume Title
Title
SERCA-phospholamban structural dynamics studied by electron paramagnetic resonance
Authors
Published Date
2013-07
Publisher
Type
Thesis or Dissertation
Abstract
Cardiac muscle function is regulated by calcium, with the sarcoplasmic reticulum (SR) serving as a calcium reservoir that releases its contents into the cytoplasm to initiate contraction. Relaxation requires that calcium be removed from the cytoplasm, which is accomplished primarily by the SR calcium-ATPase (SERCA), a transmembrane pump protein that consumes ATP to transport calcium to the SR interior, priming the muscle cell for another round of contraction. Cardiac SERCA is regulated by a second transmembrane SR protein, phospholamban (PLB), which binds and inhibits the ATPase unless phosphorylated at Ser16 by protein kinase A (PKA). The mechanism of SERCA inhibition by PLB, and the manner in which Ser16 phosphorylation reverses this inhibition remain poorly understood. The generally accepted model holds that calcium and PLB binding to SERCA are mutually exclusive, and that phosphorylation dissociates PLB to restore SERCA's calcium sensitivity. However, a number of spectroscopic studies have called this dissociation model into question, and instead suggest an alternative mechanism where PLB behaves as a subunit of SERCA, and changes its interactions with the ATPase upon phosphorylation to relieve inhibition.The work presented in this thesis tests several aspects of this alternative regulatory model, the Subunit Model, using spin-labeling and electron paramagnetic resonance (EPR) spectroscopy. In the first study, we used EPR to determine whether the inhibitory domain of PLB, its lone transmembrane helix, remains associated with SERCA following Ser16 phosphorylation. We found that the transmembrane helix of phosphorylated PLB remains bound to SERCA, in support of the Subunit Model, with our results hinting at a subtle structural change induced by phosphorylation. In the second study, we investigated the role of PLB's dynamic cytoplasmic helix in SERCA regulation, which equilibrates between an ordered T state and a dynamically disordered R state. Using charged lipids, we perturbed the electrostatic interactions between PLB's positively-charged cytoplasmic helix and the lipid bilayer, and then used EPR to resolve the T and R populations. We found that perturbation of the T/R equilibrium by charged lipids directly correlated with the functional state of SERCA, indicating that T and R correspond to inhibitory and non-inhibitory PLB conformations within the SERCA-PLB complex. In our final and ongoing study, we returned to PLB's transmembrane domain and used EPR accessibility measurements to detect conformational changes of this helix induced by Ser16 phosphorylation. Our results suggest that phosphorylation induces a subtle change in the tilt and/or rotation of PLB's transmembrane helix relative to SERCA, which may break inhibitory interactions and restore SERCA calcium sensitivity without dissociating the regulatory complex.
Description
University of Minnesota Ph.D. dissertation. July 2013. Major: Biochemistry, Molecular Bio, and Biophysics. 1 computer file (PDF); xii, 110 pages, appendix A.
Related to
Replaces
License
Collections
Series/Report Number
Funding information
Isbn identifier
Doi identifier
Previously Published Citation
Suggested citation
James, Zachary Matthew. (2013). SERCA-phospholamban structural dynamics studied by electron paramagnetic resonance. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/158436.
Content distributed via the University Digital Conservancy may be subject to additional license and use restrictions applied by the depositor. By using these files, users agree to the Terms of Use. Materials in the UDC may contain content that is disturbing and/or harmful. For more information, please see our statement on harmful content in digital repositories.