The applicant proposes a 2-AIM strategy to explore molecular mechanisms of SERCA calcium pump regulation by its major binding partner PLB.
AIM1 describes the use of fluorescence resonance energy transfer (FRET) to quantify the AFFINITY of PLB binding interactions and the STRUCTURE of the resulting protein complexes. The proposed experiments directly measure the effect of phosphorylation and mutation of PLB on its regulatory interactions. Because the fluorescent probes are genetically encoded, observations of these membrane protein interactions are being made in living cells for the first time.
AIM2 proposes to measure the PROTEIN BINDING KINETICS of the membrane protein complexes important for cardiac Ca2+ regulation. The rate of formation and dissolution of these membrane protein complexes is an important determinant of their function, but these rates are inaccessible to classical protein-protein binding kinetics methods. The applicant has invented a new optical method that can measure membrane protein subunit exchange dynamics in living cells.

Public Health Relevance

The proteins PLB and SERCA are of clinical significance because of their central role in cardiac function and disease. Disorders of these proteins are associated with heart failure, and loss or mutation of PLB in humans results in the disease """"""""dilated cardiomyopathy"""""""". Thus, PLB is a regarded as a high value therapeutic target in the treatment of heart failure, a leading cause of death in the United States.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL092321-04
Application #
8103106
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Przywara, Dennis
Project Start
2008-07-01
Project End
2013-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
4
Fiscal Year
2011
Total Cost
$334,125
Indirect Cost
Name
Loyola University Chicago
Department
Physiology
Type
Schools of Medicine
DUNS #
791277940
City
Maywood
State
IL
Country
United States
Zip Code
60153
Makarewich, Catherine A; Munir, Amir Z; Schiattarella, Gabriele G et al. (2018) The DWORF micropeptide enhances contractility and prevents heart failure in a mouse model of dilated cardiomyopathy. Elife 7:
Robia, Seth L; Young, Howard S (2018) Skin cells prefer a slower calcium pump. J Biol Chem 293:3890-3891
Raguimova, Olga N; Smolin, Nikolai; Bovo, Elisa et al. (2018) Redistribution of SERCA calcium pump conformers during intracellular calcium signaling. J Biol Chem 293:10843-10856
Zak, Taylor J; Koshman, Yevgenia E; Samarel, Allen M et al. (2017) Regulation of Focal Adhesion Kinase through a Direct Interaction with an Endogenous Inhibitor. Biochemistry 56:4722-4731
Lamichhane, Rajan; Mukherjee, Santanu; Smolin, Nikolai et al. (2017) Dynamic conformational changes in the rhesus TRIM5? dimer dictate the potency of HIV-1 restriction. Virology 500:161-168
Himes, Ryan D; Smolin, Nikolai; Kukol, Andreas et al. (2016) L30A Mutation of Phospholemman Mimics Effects of Cardiac Glycosides in Isolated Cardiomyocytes. Biochemistry 55:6196-6204
Dvornikov, Alexey V; Smolin, Nikolai; Zhang, Mengjie et al. (2016) Restrictive Cardiomyopathy Troponin I R145W Mutation Does Not Perturb Myofilament Length-dependent Activation in Human Cardiac Sarcomeres. J Biol Chem 291:21817-21828
Blackwell, Daniel J; Zak, Taylor J; Robia, Seth L (2016) Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer. Biophys J 111:1192-1202
Smolin, Nikolai; Robia, Seth L (2015) A structural mechanism for calcium transporter headpiece closure. J Phys Chem B 119:1407-15
Abrol, Neha; de Tombe, Pieter P; Robia, Seth L (2015) Acute inotropic and lusitropic effects of cardiomyopathic R9C mutation of phospholamban. J Biol Chem 290:7130-40

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