Probing cardiac calcium regulation using fluorescence spectroscopy.

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Probing cardiac calcium regulation using fluorescence spectroscopy.

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2011-06

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Calcium (Ca2+) is stored in the sarcoplasmic reticulum (SR) in both cardiac and skeletal muscle. A Ca2+ induced Ca2+ release mechanism triggers the ryanodine receptor (RyR) to release Ca2+ from the SR into the cytoplasm. This Ca2+ discharge increases the Ca2+ concentration causing the muscle to contract. RyR is regulated by calmodulin (CaM), a Ca2+ binding protein that inhibits RyR when the [Ca2+] > mM. To relax the muscle, the Sarco-endoplasmic Reticulum Calcium Adenosine Triphosphatase (SERCA), an integral membrane enzyme, pumps Ca2+ back into the SR driven by ATP hydrolysis. In cardiac tissue, SERCA is regulated by phospholamban (PLB), an integral membrane protein that inhibits SERCA at submicromolar [Ca2+]. This inhibition is relieved either by addition of micromolar Ca2+ or by phosphorylation of PLB by cAMP-dependent protein kinase A (PKA). The goal of this research was to investigate Ca2+ regulation during muscle contraction and relaxation. The major findings included: 1) two PLB variants bind tightly to SERCA, thus competing with and displacing wild-type (WT) PLB, 2) SERCA contains a novel nucleotide binding site that is not an artifact of crystallization, and 3) oxidation of specific Met residues in CaM are vital for proteasomal degradation. Using functional co-reconstitution and fluorescence resonance energy transfer (FRET), we tested the hypothesis that the loss-of-function (LOF) mutants can compete with WT-PLB to relieve SERCA inhibition. We investigated two LOF mutants, S16E (phosphorylation mimic) and L31A, for their inhibitory potency and their ability to compete with WT-PLB. Our functional studies demonstrate that SERCA co-reconstituted with mixtures of WT-PLB and LOF PLB mutants had a lower inhibitory potency compared to SERCA and WT-PLB mixtures only. FRET experiments added further support by showing that unlabeled LOF mutants lowered the FRET between donor-labeled SERCA and acceptor-labeled WT-PLB. Thus, we have provided a convenient FRET method for screening future PLB mutants for the use in gene therapy to treat heart failure. Similarly, we used another fluorescence technique, time-resolved fluorescence resonance energy transfer (TR-FRET), to investigate nucleotide binding in SERCA. Based on biochemistry and crystallography, it has been proposed that SERCA has two distinct modes of nucleotide binding. To extend this observation from the crystal to the functional sarcoplasmic reticulum membrane, we have performed TR-FRET to measure the distance between donor-labeled SERCA and the fluorescent nucleotide TNP-ADP, in the presence and absence of inhibitors. TR-FRET experiments confirmed a novel binding site in SERCA, bringing the gamma-phosphate of ADP closer to the phosphorylation site, Asp351, compared to other crystal structures with bound nucleotide. To determine whether these modes of nucleotide binding occur in solution during SERCA enzymatic cycle, we performed transient TR-FRET ([TR]2FRET) experiments, in which a complete subnanosecond TR-FRET decay was recorded every 0.1 ms after rapid mixing of donor-labeled SERCA and TNP-ADP in a stopped-flow instrument. We clearly observed a biphasic reaction with a fast component (260 s-1) and a slower component (17 s-1). TR-FRET is a powerful technique for connecting structural dynamics of SERCA with its static crystal structures. The major focus of this research has been muscle relaxation through the interaction of SERCA and PLB utilizing fluorescence spectroscopy. However, another project with implications for muscle contraction concentrated on the signals for proteasomal degradation by using CaM as a model system. CaM variants were designed using site-directed mutagenesis in order to perform site-specific oxidation of Met residues. Utilizing circular dichroism (CD), thermodynamic stability CD experiments, and proteasomal degradation assays, it was demonstrated that oxidation of Met residues 51, 71, and 72 located in the N-terminus of CaM are essential for degradation. Functional data from ryanodine binding assays showed that oxidation of Met residues in the C-terminus of CaM completely abolished CaM's ability to bind and inhibit RyR. Accumulation of these CaM within the cell could be detrimental to CaM regulation of RyR impairing Ca2+ regulation during muscle contraction.

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University of Minnesota Ph.D. dissertation. June 2011. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: David D. Thomas. 1 computer file (PDF); xvii, 114 pages, appendices I-II.

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Lockamy, Elizabeth Lee. (2011). Probing cardiac calcium regulation using fluorescence spectroscopy.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/109760.

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