Browsing by Subject "Phospholamban"

Now showing 1 - 15 of 15
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    Accurate quantitation of phospholamban expression and phosphorylation in biological samples
    (2013-06) Ablorh, Naa-Adjeley Dromoh
    Phospholamban (PLB) reversibly inhibits the sarcoplasmic reticulum calcium ATP-ase (SERCA) in cardiomyocytes. When SERCA is active, it pumps calcium into the sarcoplasmic reticulum (SR) to reduce cytosolic [Ca++]. Calcium efflux from the cytosol reduces the Ca++ available to the cytosolic contractile apparatus so that the heart can relax during diastole. The extent of relaxation depends on the amount of calcium that SERCA removes from the cytosol during diastole, while the contractile force depends on the magnitude of the end-diastolic calcium transient. Thus SERCA inhibition affects both the contractile and relaxation phases of the cardiac cycle. Unphosphorylated PLB (uPLB) inhibits SERCA at low Ca++ concentration and phosphorylated PLB (pPLB) is less inhibitory, so myocardial physiology and pathology depend critically on the mole fraction of pPLB, Xp, equal to pPLB/(uPLB+pPLB), the concentrations of total PLB (tPLB) and SERCA, and uPLB/SERCA. Prior to our assay, neither Xp nor tPLB could be measured accurately. Previous measurements relied on radioactive tracers, which only measured changes in these parameters, or immunoblots, which did not provide acceptable precision or accuracy. The fundamental problems with immunoblots were due to the lack of (a) accurate standards for pPLB and uPLB, (b) antibodies completely specific for pPLB and uPLB, and (c) a mathematical relationship between the antibody selectivity, the intensities of the samples and Xp. I have solved these problems using purified uPLB and pPLB standards, produced by solid-phase peptide synthesis, by performing two parallel immunoblots with antibodies partially specific for uPLB and pPLB, and deriving accurate equations for calculating Xp and tPLB. When this method was applied to mixtures of known composition, it measured both Xp and tPLB with ≥ 96% accuracy. I used this assay on samples of pig cardiac SR and found that Xp varied widely among four animals, from 0.08 to 0.38, but there was remarkably little variation in the ratios of Xp/tPLB and uPLB/SERCA, suggesting that PLB phosphorylation is tuned to maintain homeostasis in SERCA regulation. I have extended this method to measure accurately the mole fractions of PLB phosphorylated at Ser16, Thr17 and bisphospho-Ser16-Thr17 in biological samples, and to analyze the PLB phosphorylation status of cardiac tissue samples obtained from human patients with specific cardiomyopathies. This assay can be adapted to any phospho-protein, and with any other posttranslational modification where purified standards and partially specific antibodies are available.
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    Biophysical characterization of membrane proteins and antimicrobial peptides by solution and solid-state NMR spectroscopy.
    (2011-03) Verardi, Raffaello
    Membrane proteins and antimicrobial peptides represent two diverse and challenging classes of macromolecules to characterize at the molecular level. They are linked by the interaction with the lipid bilayer of the cell membrane. Within the lipid bilayer, membrane proteins are involved in vital biochemical processes such as ion transport, signal transduction and cell adhesion. Antimicrobial peptides are a broad class of polypeptides produced by all living organisms, representing the first line of defense against bacterial infections. They work by selectively targeting the bacterial membranes and subsequently killing the cell by a variety of mechanisms such as membrane disruption, membrane potential dissipation and enzyme inactivation. Although very important, membrane proteins and antimicrobial peptides are underrepresented in terms of available high-resolution structural information compared to water-soluble proteins and this limits the current understanding of how they work in living cells. In this thesis I summarize my contribution towards the elucidation of the high-resolution structures of the integral membrane protein phospholamban and the mechanism of action of two important antimicrobial peptides (LL37 and distinctin) by a hybrid solution and solid-state nuclear magnetic resonance spectroscopy approach. These results provide new insights and methodologies to study and understand how key membrane proteins and antimicrobial peptides elicit their function.
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    Cardiac calcium transport regulation probed by electron paramagnetic resonance spectroscopy.
    (2010-07) Torgersen, Kurt Daniel
    Muscle contraction and relaxation is regulated by calcium flux between the sarcoplasmic reticulum and the cytoplasm. Subsequent to muscle contraction, calcium must be sequestered to the sarcoplasmic reticulum in order for muscle relaxation to occur. The sarco-endoplasmic reticulum Ca-ATPase (SERCA) is a P-type ATPase embedded in the SR membrane which uses ATP hydrolysis to pump calcium back into the SR lumen to facilitate muscle relaxation. In cardiac muscle, SERCA activity is regulated by phospholamban (PLB) a 52-residue integral membrane protein which exists in a dynamic equilibrium between monomeric and pentameric species. Previous data have shown that monomeric PLB is the primary regulator of SERCA activity but recent publications have proposed that the PLB pentamer may also bind to and inhibit SERCA activity. This inhibition can be relieved by phosphorylation of PLB at Ser16, although the mechanism is not known. Electron Paramagnetic Resonance (EPR) experiments were designed to test two proposed models of the PLB pentamer, the pinwheel and bellflower. Dynamics data using the TOAC amino acid spin label showed that, like the monomer, the pentamer is in a dynamic equilibrium between ordered (T) and dynamically disordered (R) states, with the T state being predominant. Accessibility of spin labels attached to the cytoplasmic domain to the lipid bilayer showed that, like the monomer, the pentamer cytoplasmic domains strongly interact with the lipid bilayer surface. Finally, pulsed EPR (DEER) experiments measuring long range distances between spin labels attached to the cytoplasmic domain showed a bimodal distance distribution with centers at 3 and 5 nm. All of these data support the pinwheel model. To investigate SERCA binding and phosphorylation affects on PLB dynamics, a monomeric mutant, AFA-PLB, was spin labeled with TOAC either the 11 position in the cytoplasmic domain or 36 in the transmembrane domain. Conventional EPR measurements showed that phosphorylation induced and order-to-disorder conformational change in the cytoplasmic domain and that SERCA preferentially binds the PLB R state. Phosphorylation of SERCA bound PLB resulted in a disorder-to-order conformational change, suggesting that pPLB is still bound to SERCA. Conventional dynamics from 36-TOAC in the transmembrane indicated a stable helix which was unaffected by phosphorylation or SERCA binding. Dipolar EPR measurements revealed that phosphorylation of PLB in the absence of SERCA induces oligomerization and that SERCA destabilizes the pPLB oligomer. Saturation Transfer EPR data which measures the rotational diffusion of PLB in the lipid bilayer supported the conclusion that phosphorylation of PLB in the absence of SERCA induces oligomerization and showed directly that phosphorylated PLB is still bound to active SERCA. These data support the model that phosphorylation dependent relief of SERCA inhibition does not require dissociation of the SERCA-PLB complex, but is rather the result of a structural change in the complex.
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    Characterization of the conformational states of phospholamban and their roles in regulation of SR Calcium-ATPase
    (2012-12) Gustavsson, Bengt Martin
    Membrane proteins constitute 30% of the human genome but represent only a small fraction of the known three-dimensional protein structures. In this thesis I describe the characterization of the membrane protein complex between sarcoplasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLN). SERCA drives cardiac muscle relaxation by active transport of Ca2+ ions into the SR. PLN is a small membrane protein that consists of a helical trans-membrane domain connected to a cytoplasmic domain through a short loop, and inhibits SERCA through intra-membrane interactions. The cytoplasmic domain of PLN is in equilibrium between a helical, membrane-associated T state and an unfolded, membrane-dissociated R state. Here, I summarize the work to probe the structures of the T and R states and elucidate the role of the conformational equilibrium in regulation of SERCA. Using solution and solid state NMR in combination with biochemical assays I show that the structures of T and R state but not their relative populations are conserved in different lipid environments and sample conditions. Furthermore, the T/ R equilibrium has a central role in SERCA regulation and is crucial to relieve the inhibition of the enzyme. These findings provide new insights into SERCA/PLN function and offer a unique view of the role of conformational equilibria and multiple conformational states in membrane protein structure and function.
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    Examining the Role of Phospholamban Phosphorylation on Interaction with SERCA Using Fluorescence Microscopy
    (2018-07) Haydon, Suzanne
    Regulation of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) by Phospholamban (PLB) plays a crucial role in normal cardiomyocyte function through controlling the speed and extent of myocyte relaxation. The interaction between PLB and SERCA is altered in many forms of heart failure (HF), making these proteins potential targets for the treatment of HF. Both proteins have been extensively studied in vitro, where their basic structure and function were determined, and in animal models, where their role in disease was examined. However, key information connecting the in vitro experiments and animal models is needed to better understand the PLB-SERCA interaction and to design effective HF treatment strategies. In particular, we wanted to examine two conflicting in vitro models of how the PLB-SERCA interaction changes after PLB phosphorylation: the dissociation model and the structural model. In the dissociation model, phosphorylation causes PLB to dissociate from SERCA, while in the structural model, phosphorylation causes a shift in the PLB binding position along SERCA. In order to determine the correct model of PLB-SERCA interaction in live cells, we expressed cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to the N-termini of SERCA and PLB respectively, in HEK293 cells for fluorescence resonance energy transfer (FRET) microscopy experiments. We were able to use the native beta-adrenergic signaling system in the cells to control the state of PLB phosphorylation in a time-dependent manner. For the dissociation model to be true, we expected to see a significant reduction in FRET between CFP-SERCA and YFP-PLB after PLB phosphorylation. While significant increases in PLB phosphorylation were produced in the cells, FRET did not decrease. Instead, FRET increased with PLB phosphorylation at serine 16, indicating either a shorter distance between PLB and SERCA, or higher binding of PLB to SERCA. As the beta-adrenergic signal progressed through the cells, causing phosphorylation of PLB at threonine 17, FRET returned towards basal levels, but did not show the decrease below basal FRET levels that would indicate PLB dissociation from SERCA. Thus, we determined that there is a subtle change in the PLB-SERCA interaction due to PLB phosphorylation rather than a large scale dissociation. In order to differentiate changes in distance from changes in binding time-resolved (TR)-FRET experiments were required. Fluorescence lifetime imaging microscopy (FLIM) is a variant of TR-FRET that measures fluorescence decay curves with a fast-pulsed laser and photon counting board attached to a confocal microscope. These fluorescence decay curves provide more information than intensity measurements since they can be fit to multiple exponentials to test different interaction models. FLIM is a relatively new technique, thus we worked on developing appropriate experimental conditions for acquiring fluorescence decays that contained enough photons for multi-exponential fitting while still measuring individual cells. We were able to use FLIM to measure FRET values similar to those acquired on standard fluorescence microscopes and confirmed that phosphorylation of PLB did not cause dissociation from SERCA. However, further improvements to FLIM acquisition and analysis are needed for the multi-exponential fitting to provide a better model of PLB-SERCA interaction in live cells.
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    Fluorescence tools to identify Novel SERCA activators
    (2013-08) Gruber, Simon Joseph
    One of the universal hallmarks of heart failure is defective calcium cycling. The calcium concentration in a muscle cell must be high to cause contraction and low to allow relaxation, and most of the calcium removal is accomplished by the intracellular membrane pump known as the sarco-endoplasmic reticulum calcium ATPase (SERCA). When SERCA activity is too low in cardiac muscle, the heart does not fully relax and fill with blood, so the next contraction cannot pump enough blood through the body. The ubiquity of calcium cycling dysfunction in heart failure and other muscle diseases has made SERCA a major target for novel heart failure therapeutics since the late 1990s. All of the work presented in this thesis focuses on methods to activate SERCA as a treatment for heart failure. SERCA is regulated by phospholamban (PLB) in heart muscle, preventing the enzyme from being fully active all the time but allowing maximal activity when the body demands. Some methods of activating SERCA seek to remove the inhibitory effects of PLB, either partially or fully. In this thesis, PLB mutants are investigated as potential gene therapy vectors. PLB mutants that are less inhibitory but still bind to SERCA could allow the enzyme to be more active if they displace endogenous PLB. A FRET assay using genetically engineered fluorescent fusions of SERCA and PLB expressed stably in a human cell line was used to measure the ability of different mutants to compete for SERCA binding. Fluorescently labeled SERCA and PLB were also reconstituted in an in vitro lipid bilayer system to screen for small-molecule compounds that activate SERCA. Several compounds were found to decrease SERCA-PLB FRET and many of these turned out to be SERCA activators that improved myocyte contractility. However, none of the compounds were specific to the SERCA-PLB interaction. Finally, an intramolecular FRET assay was developed to detect changes in the relative distance between cytoplasmic domains within SERCA in living cells. This assay was used to screen a small-scale compound library to show that FRET between SERCA domains is sensitive to both activators and inhibitors of SERCA function. All of these FRET assays are being followed up in the Thomas lab to identify potential SERCA activators for heart failure and other diseases.
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    Mechanism of Phospholamban Activation by HAX-1 and their Roles in the Regulation of the SR Calcium-ATPase
    (2021-04) Larsen, Erik
    The Ca2+ transient of the cardiomyocyte is key to the contractility of the heart. Its dysregulation has been associated with heart disease, leading to investigation of the regulation of Ca2+ for potential drug targets. The Sarcoplasmic Endo-Reticulum Ca2+ ATP-ase (SERCA) pump and its main inhibitor in heart tissue, phospholamban (PLN), are two promising targets that are under β-adrenergic control via phosphorylation of PLN by Protein Kinase A (PKA). Phosphorylation of Ser16 on the cytoplasmic domain of PLN results in decreased inhibition of SERCA. Recently, an additional member of the SERCA interactome has been discovered called Hematopoietic lineage cell-specific protein 1 (HCLS1) Associated Protein X-1 (HAX-1). Contrasting PLN phosphorylation, the interaction of HAX-1 and PLN increases the inhibition of PLN for SERCA, adding another layer of complexity to SERCA regulation and a potential new drug target. This thesis aims to investigate the structure-function relationship of the ternary complex, SERCA/PLN/HAX-1 using NMR spectroscopy as the primary technique.
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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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    Probing cardiac calcium regulation using time-resolved optical spectroscopy.
    (2012-07) Li, Ji
    Sarcoplasmic reticulum calcium adenosine triphosphatase (SERCA) is an integral membrane Ca2+ pump that reuptakes Ca2+ into the lumen of sarcoplasmic reticulum (SR), decreases the cytoplasmic Ca2+ concentration, and initiates relaxation in both skeletal and cardiac muscle. In the heart, SERCA activity is partially inhibited by phospholamban, another integral membrane protein. This inhibition can be relieved by high Ca2+ concentration or phosphorylation of PLB. Misregulation of Ca2+ by the SERCA-PLB complex is associated with heart failure. The goal of this thesis is to investigate the mechanism of Ca2+ regulation by the SERCA-PLB complex, by direct measurement of their physical interactions using optical spectroscopy. We have used time-resolved fluorescence resonance energy transfer (TRFRET) to determine the binding and distance distribution between the donor-labeled SERCA and acceptor-labeled SERCA-bound PLB in artificial membranes. Results show that PLB binds to SERCA with two structures, an extended R state with the cytoplasmic domain pointing away from the membrane surface, resulting in a shorter interprobe distance, and a T state with the cytoplasmic domain bound to the membrane, giving rise to a longer interprobe distance. We modulated the electrostatic interaction between SERCA and PLB by adjusting the membrane headgroup charge. Results show that the T state of PLB is more inhibitory than R. SERCA inhibition by PLB is well correlated with the T/R equilibration and does not require dissociation of the complex. We used time-resolved phosphorescence anisotropy (TPA) to analyze the functional oligomeric regulation of SERCA by Ca2+ and PLB phosphorylation in native cardiac SR. A uniaxial rotation diffusion analysis of TPA data was used to resolve the oligomeric state of SERCA. We found that SERCA, in both the skeletal and cardiac SR, consists of 3 oligomeric species plus an immobilized large aggregate. SERCA activation by either Ca2+ or PLB phosphorylation correlates with the destabilization of the immobilized large aggregate, without significantly affecting the sizes of the 3 oligomeric states. In summary, PLB regulates SERCA through two independent mechanisms. In the bound SERCA-PLB complex, the structural dynamics of PLB determines its inhibitory effect on SERCA.. In addition, Ca2+ and phosphorylation of PLB regulate SERCA activity by dissociating the immobilized SERCA aggregates and increasing the functional oligomeric states.
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    Rational design of loss-of-function phospholamban mutants to tune SERCA function.
    (2012-04) Ha, Kim N.
    Unphosphorylated phospholamban (PLN) is the endogenous inhibitor of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA), the enzyme that regulates cardiac muscle relaxation in humans. In its phosphorylated state, PLN (pS16-PLN, pT17-PLN, and pS16pT17-PLN) does not inhibit SERCA. Dysfunctions in SERCA:PLN interactions and in the PLN phosphorylation mechanism have been implicated in cardiac disease and targeting PLN is becoming a viable avenue for treating heart disease. Specifically, innovative genetic treatments using recombinant adeno-associated virus (rAAV) with S16E-PLN, a pseudo-phosphorylated form of PLN, have shown a remarkable efficacy in reducing the progression of cardiac failure in both small and large animals. The following thesis summarizes efforts to rationally design PLN mutants to tune SERCA function. Using a combination of NMR spectroscopy and biochemical assays, we have built a structure-dynamics-function correlation that shows PLN can be tuned to augment SERCA function by acting on the conformational coupling between the cytoplasmic and transmembrane domain and by pseudo-phosphorylation. Additionally, to better understand the role of mutation in PLN:SERCA interactions, we also investigated a mutant of PLN (R9C) known to be linked to hereditary dilated cardiomyopathy, showing that the mutation disrupts the pentamer-monomer equilibrium, and that these effects are exacerbated under oxidizing conditions. Insights to these issues will provide better paradigms with which to design therapeutic mutants of PLN for treatment of heart failure.
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    SERCA-phospholamban structural dynamics studied by electron paramagnetic resonance
    (2013-07) James, Zachary Matthew
    Cardiac muscle function is regulated by calcium, with the sarcoplasmic reticulum (SR) serving as a calcium reservoir that releases its contents into the cytoplasm to initiate contraction. Relaxation requires that calcium be removed from the cytoplasm, which is accomplished primarily by the SR calcium-ATPase (SERCA), a transmembrane pump protein that consumes ATP to transport calcium to the SR interior, priming the muscle cell for another round of contraction. Cardiac SERCA is regulated by a second transmembrane SR protein, phospholamban (PLB), which binds and inhibits the ATPase unless phosphorylated at Ser16 by protein kinase A (PKA). The mechanism of SERCA inhibition by PLB, and the manner in which Ser16 phosphorylation reverses this inhibition remain poorly understood. The generally accepted model holds that calcium and PLB binding to SERCA are mutually exclusive, and that phosphorylation dissociates PLB to restore SERCA's calcium sensitivity. However, a number of spectroscopic studies have called this dissociation model into question, and instead suggest an alternative mechanism where PLB behaves as a subunit of SERCA, and changes its interactions with the ATPase upon phosphorylation to relieve inhibition.The work presented in this thesis tests several aspects of this alternative regulatory model, the Subunit Model, using spin-labeling and electron paramagnetic resonance (EPR) spectroscopy. In the first study, we used EPR to determine whether the inhibitory domain of PLB, its lone transmembrane helix, remains associated with SERCA following Ser16 phosphorylation. We found that the transmembrane helix of phosphorylated PLB remains bound to SERCA, in support of the Subunit Model, with our results hinting at a subtle structural change induced by phosphorylation. In the second study, we investigated the role of PLB's dynamic cytoplasmic helix in SERCA regulation, which equilibrates between an ordered T state and a dynamically disordered R state. Using charged lipids, we perturbed the electrostatic interactions between PLB's positively-charged cytoplasmic helix and the lipid bilayer, and then used EPR to resolve the T and R populations. We found that perturbation of the T/R equilibrium by charged lipids directly correlated with the functional state of SERCA, indicating that T and R correspond to inhibitory and non-inhibitory PLB conformations within the SERCA-PLB complex. In our final and ongoing study, we returned to PLB's transmembrane domain and used EPR accessibility measurements to detect conformational changes of this helix induced by Ser16 phosphorylation. Our results suggest that phosphorylation induces a subtle change in the tilt and/or rotation of PLB's transmembrane helix relative to SERCA, which may break inhibitory interactions and restore SERCA calcium sensitivity without dissociating the regulatory complex.
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    Spectroscopic function-structure analysis of the SERCA-PLB complex
    (2014-06) Dong, Xiaoqiong
    Cardiomyocyte contraction is controlled by intracellular Ca2+ concentrations. Action potential opens the voltage-gated calcium channel in the sarcolemma and triggers the calcium-induced calcium-release mechanism to release Ca2+ stored in the sarcoplasmic reticulum (SR) through ryanodine receptors. For muscle relaxation to occur, Ca2+ must be removed from the cytosol. Most of the activator Ca2+ is sequestered back into the SR by sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). In ventricular myocytes, SERCA is regulated by a small integral membrane protein, phospholamban (PLB). PLB binds and inhibits SERCA, and this inhibition is physiologically relieved by either micromolar Ca2+ in systole, or by phosphorylation at Ser16 or Thr17 through β-adrenergic stimulation. A decline in SERCA activity is implicated in heart failure irrespective of etiologies. Recent gene therapies for heart failure emphasize enhancing SERCA activity by decreasing PLB inhibition. However, the structural mechanism of relief of inhibition still remains elusive. This thesis work is motivated to elucidate the structural basis for SERCA regulation by PLB, hence providing more information for the design of next generation gene and drug therapies.This thesis work uses time-resolved fluorescence resonance energy transfer (TR-FRET) to probe the structures of the SERCA-PLB complex in its activated or inhibited forms. In the first project, we investigated the function effect of the equilibrium of PLB cytoplasmic domain between an ordered R state and a disordered T state on SERCA regulation. We varied the lipid headgroup charges to perturb this equilibrium through electrostatic interactions with the positively charged PLB cytoplasmic domain. TR-FRET measurements, in conjunction with functional data and electron paramagnetic resonance experiments, established the correlation of the T/R equilibrium with PLB inhibitory potency. In the second project, we studied the structures of the SERCA-PLB complex under physiological conditions that relieve inhibition. TR-FRET distance measurements between cytoplasmic domains of SERCA and PLB revealed that phosphorylation of PLB at Ser16 relieves SERCA inhibition mainly by shifting the T/R equilibrium toward the less inhibitory R state, and partially by dissociating the complex. Micromolar Ca2+ probably relieves inhibition through structural rearrangements within the transmembrane domain of the complex. In the last project discussed in this thesis, we used western blot to quantify different phosphorylation states of PLB in pig cardiac SR. PLB can be phosphorylated at either Ser16 or Thr17, generating four phosphorylation states: unphosphorylated, phosphorylated only at Ser16, phosphorylated only at Thr17, or phosphorylated at both sites. We also found that each PLB phosphorylation state has a distinct inhibitory potency for SERCA.
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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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    Structural Characterization of Sarcolipin by Solid State NMR and Investigation of its Role in the Regulation of Sarco(endo)plasmic Reticulum Calcium Adenosine-Triphospatase
    (2014-02) Mote, Kaustubh
    Structural characterization of membrane proteins and their complexes is an important and ever growing challenge to the classical techniques of biomolecular structural characterization. Rapid developments in the field of solid state NMR (ssNMR) have opened up an exciting new alternative to X-ray crystallography, as these studies can now be performed in fully hydrated lipid bilayers that faithfully mimic the physiologically relevant conditions. Nonetheless, routine application of ssNMR on biomolecular systems is hampered by their low sensitivity and spectral resolution. In this work, we have addressed these challenges by developing new strategies to study membrane proteins by ssNMR. With a set of improved pulse sequences for oriented and magic angle spinning techniques in ssNMR, we determined the topology (i.e. the structure and transmembrane orientation) of sarcolipin, a regulator of the Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA), in lipid bilayers. These techniques are further used to study the complex between these two proteins and understand the molecular basis for this regulatory interaction. The methodological developments reported here are transferable to studies on other membrane proteins and they clear several roadblocks in the successful application of ssNMR for these challenging bio-molecular systems. Finally, we present how these studies have furthered our understanding of the regulation of muscle relaxation process by SERCA. These findings represent the first steps in designing new therapeutic approaches for cardiac and skeletal muscle disorders.
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    Tuning the Equilibrium: A Biophysical Approach to Controlling Cardiac Contractility through SERCA and Phospholamban
    (2017-05) Soller, Kailey
    Heart disease is the leading cause of death throughout the world and one of the major hallmarks is dysfunctional muscle contractility. Contractility is a highly regulated and complex process which involves multiple proteins. This network can be easily disrupted by mutation or changes in protein expression level and thus, proteins involved in this process are key drug targets. Muscle contractility is controlled by the calcium concentration in the cytosol. In cardiomyocytes, the sarco(endo)plasmic reticulum Ca2+ -ATPase, SERCA, and its regulatory protein, phospholamban (PLN) are responsible for ~70% of Ca2+ reuptake into the SR. While unphosphorylated, PLN inhibits SERCA by lowering its apparent Ca2+ affinity. Upon phosphorylation by PKA at Ser16, PLN inhibition is relieved. Mutations or disruptions in this complex have found in many forms of heart disease; thus, understanding the molecular interactions between SERCA and PLN, along with possible regulatory molecules, is essential. Understanding the molecular mechanisms that occur on a beat-to-beat basis will be essential for developing therapeutics to treat cardiomyopathies. In this dissertation work, I studied the structural, biochemical and biophysical properties of SERCA and PLN. We found that single-stranded DNA (ssDNA), RNA, and DNA analogs bind the cytoplasmic domain of PLN with low nanomolar dissociation constants, relieving inhibition of SERCA. The relief of inhibition is length-dependent, while affinity is constant for oligonucleotides longer than 10 bases. Solution and solid-state NMR experiments have provided residue specific information that ssDNA targets the cytoplasmic domain of PLN and does not affect SERCA in the absence of PLN. In-cell FRET and NMR experiments determined that addition of ssDNA does not dissociate PLN from SERCA. Additionally, I started moving this work into a sustainable, in vivo system using cardiomyocytes derived from induced pluripotent stem cells (iPSCs) to investigate the functional effects of these molecules with PLN in cell. The establishment of this cell line in our laboratory will allow for future characterization of not only XNAs with PLN, but also experiments with any cell type obtainable through differentiation of iPSCs. Finally, early work with the R14del hereditary mutant of PLN helped to determine the structural changes this mutant imposes on PLN as well as when in complex with SERCA. We found that the R14del mutant is loss-of-function and also if phosphorylated, will not relieve inhibition of SERCA. We believe that the knowledge gained here on the SERCA-PLN complex contributes to the overall understanding of how calcium handling can be modulated to change protein function and demonstrates a novel avenue of oligonucleotide action in the body. miRNAs may have evolved to also directly interact with non-transcription related proteins to modulate their function. These results and our future experiments will provide a promising avenue for development of novel therapeutic regulators of the SERCA-PLN complex to help treat heart disease, as well as provide some of the needed mechanistic insight on how hereditary mutants like R14del cause aberrant regulation in the heart.