The overall goal of the proposal is to understand calcium regulation in both cardiac and skeletal muscle at the atomic level. Calcium is an essential messenger for muscle contractility and its homeostatic balance is controlled by proteins embedded in the sarcoplasmic reticulum (SR) membrane. In cardiac myocytes, the SR Ca2+-ATPase 2a isoform (SERCA2a) is responsible for ~70% of the Ca2+ translocation and regulates diastole. SERCA2a is inhibited by phospholamban (PLN), a membrane protein that reverses its inhibition upon phosphorylation at Ser16 and Thr17. In skeletal muscle, the SERCA1a isoform administrates the relaxation phase and is regulated by sarcolipin (SLN), a membrane inhibitor that is post-translationally regulated by phosphorylation at Thr5. Both PLN and SLN maintain SERCA's activity within a physiological window of apparent Ca2+ affinity. When SERCA2a functions outside this window, disruptions in Ca2+ homeostasis leads to dilated or hypertrophic cardiomyopathies, and ultimately heart failure. SERCA1a dysfunctions result in reduced skeletal contractility, leading to conditions such as Brody disease. In past funding cycles, we characterized the structural dynamics of both PLN and SLN in the presence and absence of the ATPase. The latter enabled us to design and test new dominant-negative PLN mutants with promising results towards improving muscle contractility via rAAV-mediated gene therapy. In this competitive renewal, we propose to analyze the effects of the phosphorylation states in both PLN and SLN through investigating the interactions between these two inhibitors and the enzyme along the catalytic cycle, and take advantage of this knowledge in designing mutants with improved loss-of-function characteristics. To carry out these studies, we will utilize a combination of molecular biology, biochemical assay, as well as spectroscopic methods (NMR, EPR, and fluorescence) that will enable the analysis of these membrane protein complexes in native lipids.

Public Health Relevance

The proposal focuses on the structural analysis of the interactions between the sarcoplasmic reticulum Ca2+-ATPase and its two inhibitors, phospholamban and sarcolipin. Correct functioning of these of these protein complexes is directly linked with cardiac and skeletal muscle diseases, such as dilated cardiomyopathy, hypertrophy, and Brody disease. Understanding the structural details of these interactions will be instrumental to designing innovative therapies to treat these devastating diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM064742-12
Application #
8636025
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Wehrle, Janna P
Project Start
2002-09-16
Project End
2016-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
12
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Chipot, Christophe; Dehez, François; Schnell, Jason R et al. (2018) Perturbations of Native Membrane Protein Structure in Alkyl Phosphocholine Detergents: A Critical Assessment of NMR and Biophysical Studies. Chem Rev 118:3559-3607
Nelson, Sarah E D; Ha, Kim N; Gopinath, Tata et al. (2018) Effects of the Arg9Cys and Arg25Cys mutations on phospholamban's conformational equilibrium in membrane bilayers. Biochim Biophys Acta Biomembr 1860:1335-1341
Wang, Songlin; Gopinath, T; Veglia, Gianluigi (2018) Application of paramagnetic relaxation enhancements to accelerate the acquisition of 2D and 3D solid-state NMR spectra of oriented membrane proteins. Methods 138-139:54-61
Harmouche, Nicole; Aisenbrey, Christopher; Porcelli, Fernando et al. (2017) Solution and Solid-State Nuclear Magnetic Resonance Structural Investigations of the Antimicrobial Designer Peptide GL13K in Membranes. Biochemistry 56:4269-4278
Gopinath, T; Nelson, Sarah E D; Veglia, Gianluigi (2017) 1H-detected MAS solid-state NMR experiments enable the simultaneous mapping of rigid and dynamic domains of membrane proteins. J Magn Reson 285:101-107
Manu, V S; Veglia, Gianluigi (2016) Optimization of identity operation in NMR spectroscopy via genetic algorithm: Application to the TEDOR experiment. J Magn Reson 273:40-46
Gopinath, T; Veglia, Gianluigi (2016) Multiple acquisitions via sequential transfer of orphan spin polarization (MAeSTOSO): How far can we push residual spin polarization in solid-state NMR? J Magn Reson 267:1-8
Sanz-Hernández, Máximo; Vostrikov, Vitaly V; Veglia, Gianluigi et al. (2016) Accurate Determination of Conformational Transitions in Oligomeric Membrane Proteins. Sci Rep 6:23063
Dicke, Alysha; Gopinath, Tata; Wang, Yingjie et al. (2016) Probing Residue-Specific Water-Protein Interactions in Oriented Lipid Membranes via Solid-State NMR Spectroscopy. J Phys Chem B :
Vostrikov, Vitaly V; Gustavsson, Martin; Gopinath, Tata et al. (2016) Ca(2+) ATPase Conformational Transitions in Lipid Bilayers Mapped by Site-directed Ethylation and Solid-State NMR. ACS Chem Biol 11:329-34

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