The overall goal of this research is to elucidate the structural basis of dilated cardiomyopathy (DCM). Cardiac muscle function depends critically on Ca-ATPase, a membrane-embedded Ca transport enzyme inhibited by phospholamban (PLB). Upon adrenergic stimulation, this inhibition is reversed by cAMP-dependent protein kinase (PKA), phosphorylating PLB at S16. Disruptions to this system result in heart disease. A specific PLB mutation (R9C-PLB) has been linked directly to hereditary DCM. R9C-PLB irreversibly inhibits PKA, hampering the phosphorylation of wt-PLB, interfering with Ca transport regulation, and causing heart failure. This finding represents a breakthrough that has been widely reported in the national media. We will determine the structural basis of DCM using molecular biology, peptide synthesis, NMR, and EPR methods to accomplish the following AIMs: ? ? AIM 1: Characterizing the phosphorylation kinetics of both PLB and its lethal R9C mutant. ? ? AIM 2: Probing the binding surfaces and the structures of PKA/PLB and PKA/R9C-PLB complexes. ? ? AIM 3: Mapping the structural dynamics of PKA and PKA complexes with PLB and its R9C-mutant. ? ? AIM 4: Elucidating the membrane architecture of the PKA/PLB complex in lipids: influence of N-myristoylation of PKA. ? ? The immediate outcome of this research will be to characterize the high-resolution structure of the R9C-PLB/PKA complex and to elucidate the structural links between R9C mutation and DCM. The long-term goal is to develop a molecular framework for new therapeutic approaches involving calcium regulation and heart muscle contractility. To the best of our knowledge, these are the first biophysical studies of protein-protein interactions between integral membrane proteins and soluble proteins ever carried out using NMR spectroscopy. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM072701-02S1
Application #
7680969
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Chin, Jean
Project Start
2007-06-01
Project End
2011-05-31
Budget Start
2008-06-01
Budget End
2009-05-31
Support Year
2
Fiscal Year
2008
Total Cost
$68,398
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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
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
Kim, Jonggul; Ahuja, Lalima G; Chao, Fa-An et al. (2017) A dynamic hydrophobic core orchestrates allostery in protein kinases. Sci Adv 3:e1600663
Kim, Jonggul; Wang, Yingjie; Li, Geoffrey et al. (2016) A Semiautomated Assignment Protocol for Methyl Group Side Chains in Large Proteins. Methods Enzymol 566:35-57
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
Kim, Jonggul; Li, Geoffrey; Walters, Michael A et al. (2016) Uncoupling Catalytic and Binding Functions in the Cyclic AMP-Dependent Protein Kinase A. Structure 24:353-63
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 :
Soller, Kailey J; Yang, Jing; Veglia, Gianluigi et al. (2016) Reversal of Phospholamban Inhibition of the Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA) Using Short, Protein-interacting RNAs and Oligonucleotide Analogs. J Biol Chem 291:21510-21518
Vostrikov, Vitaly V; Soller, Kailey J; Ha, Kim N et al. (2015) Effects of naturally occurring arginine 14 deletion on phospholamban conformational dynamics and membrane interactions. Biochim Biophys Acta 1848:315-22

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