The overall goal of the proposed research is to determine the structural basis of Ca-ATPase (calcium pump) regulation mechanisms in both skeletal and cardiac sarcoplasmic reticulum (SR). Calcium is the carrier of contractile stimuli in mammalian organisms. Its homeostatic balance is maintained by Ca-ATPase, a 110 kDa integral membrane protein. There are two small integral membrane proteins that regulate this pump: Phospholamban, a 52-amino-acid phosphoprotein expressed in cardiac muscle, and sarcolipin, a 31-amino-acid protein expressed in skeletal muscle. The goals of this research are to determine the structures of these two proteins in detergent and lipid environments, to determine their interactions with the Ca-ATPase, and to evaluate the structural effects of functionally important modifications, such as mutation and phosphorylation. A multidisciplinary approach will be used, involving solid-phase peptide synthesis, cell culture, site-directed mutagenesis, protein expression and purification, membrane reconstitution, enzyme kinetics, and a combination of solution and solid-state NMR techniques. The following specific aims will be pursued:
AIM 1 : NMR structural determination of phosphorylated and unphosphorylated forms of phospholamban in detergent micelles.
AIM 2 : NMR structural determination of sarcolipin and its variants in detergent micelles.
AIM 3 : Evaluation of the interactions of both phospholamban and sarcolipin with Ca-ATPase in detergent micelles.
AIM 4 : Solid-state NMR determination of the orientations of both phospholamban and sarcolipin in lipid bilayers and their interactions with the Ca-ATPase.The outcomes of this research will be complemented by the structural and dynamics information from site-directed spin and fluorescence labeling studies obtained by our collaborator, Dr. Thomas.Taken with the recently determined high-resolution structure of the Ca-ATPase pump (SERCA1 a) (Toyoshima et al., 2000), the structures of sarcolipin and phospholamban and their interactions with the Ca-ATPase will provide the molecular framework needed to understand calcium transport regulation and its role in muscle function and malfunction. On a more general level, we hope that the approach being developed here - to use a combination of biological and biophysical techniques to probe the functional and structural dynamics of protein-protein interactions within the membrane plane - will have wide application to other important problems in membrane biophysics and physiology.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Wehrle, Janna P
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Minnesota Twin Cities
Schools of Arts and Sciences
United States
Zip Code
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
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
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
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

Showing the most recent 10 out of 80 publications