Browsing by Subject "Calmodulin"

Now showing 1 - 3 of 3
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    Probing cardiac calcium regulation using fluorescence spectroscopy.
    (2011-06) Lockamy, Elizabeth Lee
    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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    Structural and dynamics analysis of pathogenic modifications in cardiac sarcoplasmic reticulum proteins involved in Ca2+ transport
    (2018-01) Nelson, Sarah
    Calcium signaling pathways are essential for the coordination of contraction and relaxation in cardiac muscle. Disruption of cardiac calcium cycling by pathogenic modifications in calcium transport proteins leads to a variety of cardiomyopathies including dilated cardiomyopathy and arrhythmias. The following thesis summarizes the structural and dynamic characterization of key regulatory proteins involved in calcium release and reuptake in the sarcoplasmic reticulum (SR). Calmodulin (CaM), a calcium-sensing protein that regulates its cellular targets based on the level of calcium in the cell, mediates calcium release from the SR via the homotetrameric calcium release channel, ryanodine receptor (RyR). The CaM-RyR complex has been a challenging structural target due to the size and complexity of the RyR. By applying a combination of solution and solid-state NMR techniques we have begun to develop a molecular model for CaM’s regulation of the RyR and how this regulation is disrupted by pathogenic modifications such as oxidation and mutation. Disruptions in calcium reuptake to the SR due to mutations in the small transmembrane protein, phospholamban (PLN), result from dysregulation of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). These PLN mutations are primarily associated with the development of dilated cardiomyopathy and by applying solution and solid-state NMR techniques we have begun to develop a model for how changes in PLN’s structure and dynamics correlate to the dysregulation of SERCA. Together, the structural and dynamic studies outlined in this thesis provide further insights into the correlations between protein structure and function and the crucial roles CaM and PLN play in cardiac function.
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    Transcriptional and functional study of Arabidopsis defence response against Pseudomanas syringae.
    (2009-01) Wang, Lin
    Using a reverse genetic approach, we investigated genes that are potentially important for disease resistance against the bacterial pathogen of Arabidopsis thaliana: Pseudomonas syringae. Genes that were induced at least two-fold after infection by Pseudomonas syringae pv. maculicola ES4326 were chosen as candidates for our study. Arabidopsis T-DNA mutants were ordered and assayed for bacterial growth. Mutants that consistently supported more bacterial growth than wild type controls were selected for further analysis. We also monitored expression profiles of wild-type plants and mutants with defects in key components of the defense signaling network using a microarray. The data were used to model the Arabidopsis defense network 24 hours after infection by Pseudomonas syringae pv. maculicola strain Psm ES4326. From the identified novel genes that are likely important for plant defense, I chose two members from the Arabidopsis CBP60 family, CBP60g and CBP60h, for functional analyses. Mutants of CBP60g and CBP60h are more susceptible to bacterial infection than wild type. They accumulated less SA in response to MAMP (Microbe Associated Molecular Pattern) and/or pathogen inoculations. CBP60g binds to calmodulin and the calmodulin binding is important to its function in disease resistance and SA signaling. In contrast, CBP60h does not bind calmodulin and seems to function independently of calcium signaling. A cbp60g and cbp60h double mutant is highly susceptible to Pseudomonas syringae infection; it is more susceptible than sid2 and comparable to pad4. It is likely that CBP60g and CBP60h share partially redundant and crucial functions in defense signaling. The cbp60g and cbp60h double mutant was also found to affect both SA-dependent and independent signaling pathways