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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Wehrle, Janna P
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University of Minnesota Twin Cities
Schools of Medicine
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Sanz-Hernández, Máximo; Vostrikov, Vitaly V; Veglia, Gianluigi et al. (2016) Accurate Determination of Conformational Transitions in Oligomeric Membrane Proteins. Sci Rep 6:23063
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
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
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Soller, Kailey J; Verardi, Raffaello; Jing, Meng et al. (2015) Rheostatic Regulation of the SERCA/Phospholamban Membrane Protein Complex Using Non-Coding RNA and Single-Stranded DNA oligonucleotides. Sci Rep 5:13000
Gopinath, T; Mote, Kaustubh R; Veglia, Gianluigi (2015) Simultaneous acquisition of 2D and 3D solid-state NMR experiments for sequential assignment of oriented membrane protein samples. J Biomol NMR 62:53-61
Manu, V S; Veglia, Gianluigi (2015) Genetic algorithm optimized triply compensated pulses in NMR spectroscopy. J Magn Reson 260:136-43
Gopinath, T; Veglia, Gianluigi (2015) Multiple acquisition of magic angle spinning solid-state NMR experiments using one receiver: application to microcrystalline and membrane protein preparations. J Magn Reson 253:143-53
Jing, Meng; Verardi, Raffaello; Veglia, Gianluigi et al. (2014) Development of a Sensitive Assay for SERCA Activity Using FRET Detection of ADP. Anal Methods 6:1468-1474
De Simone, Alfonso; Mote, Kaustubh R; Veglia, Gianluigi (2014) Structural dynamics and conformational equilibria of SERCA regulatory proteins in membranes by solid-state NMR restrained simulations. Biophys J 106:2566-76

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