Browsing by Subject "FRET"

Now showing 1 - 15 of 15
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    Autoregulation Of G Protein-Coupled Receptor Signaling Through The Third Intracellular Loop
    (2023-04) Sadler, Fredrik
    The third intracellular loop (ICL3) of the G protein-coupled receptor (GPCR) fold is important for the signal transduction process downstream of receptor activation. Despite this, ICL3’s lack of defined structure, combined with its high sequence divergence among GPCRs, obfuscates characterization of its involvement in receptor signaling. Previous studies focusing on the β2 adrenergic receptor (β2AR) suggest that ICL3 is involved in the structural process of receptor activation and signaling. We derive mechanistic insights into ICL3s role in β2AR signaling, finding that ICL3 autoregulates receptor activity through a dynamic conformational equilibrium between states that block or expose the receptor’s G protein binding site. We demonstrate the importance of this equilibrium for receptor pharmacology, finding that G protein-mimetic effectors bias ICL3’s exposed states to allosterically activate the receptor. Our findings additionally reveal that ICL3 tunes signaling specificity by inhibiting receptor coupling to G protein subtypes that weakly couple to the receptor. Despite the sequence diversity of ICL3, we demonstrate that this negative G protein selection mechanism through ICL3 extends to GPCRs across the superfamily, expanding upon the framework for how receptors mediate G protein subtype selective signaling. Furthermore, our collective findings motivate ICL3 as an allosteric site for receptor and signaling pathway specific ligands.
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    Development of High-Throughput and High-Content Analysis Assays for Neurodegeneration-Related Intrinsically Disorderd Proteins
    (2020) Nathan, Noah
    We developed a FRET-based protein-protein biosensor of Fused in Sarcoma (FUS), an Amyotrophic Lateral Sclerosis and Frontotemporal Dementia related protein. The FUS biosensor had a robust signal, yielding a FRET efficiency of 7.61% with low signal to noise ratio. Based on high-throughput FRET measurements, we determined a standard deviation of 0.0158 ns for the donor/acceptor fluorescent lifetime. In future drug screens for compounds that modulate FUS aggregation, the threshold for hits will be set at 2.45 ± 0.0474 ns (3 SD). In addition, we implemented a MATLAB script that quantifies the ratio of cytoplasmic to nuclear FUS-rich stress granules from fluorescent images of FUS-GFP-expressing N2a cells. We showed that sorbitol, which has been shown to cause FUS mislocalization via hypertonic stress, caused a shift in the cytoplasmic to nuclear ratio of FUS as compared to untreated cells. We implemented a second MATLAB automated algorithm that quantifies total neurite outgrowth from neurospheres expressing the Parkinson's disease-related protein alpha-synuclein. We found that the mutant aSyn A53T caused a reduction of 41% in total neurites as compared to WT aSyn-expressing neurospheres. This result was validated by counting neurites manually with ImageJ, which yielded a reduction in neurites of 49%.
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    Discovery of small molecule modulators of protein-protein interactions by FRET-based high-throughput screening and structure-based drug design
    (2019-08) Lo, Chih Hung
    Protein–protein interactions (PPIs) are of pivotal importance in the regulation of biological systems and are consequently implicated in the development of disease states. Here, we investigated two classes of protein, including a transmembrane protein (tumor necrosis factor receptor 1 (TNFR1)) and intrinsically disordered proteins (tau and huntingtin (HTT)), which are implicated in autoimmune diseases and neurodegenerative diseases respectively. Receptor-specific inhibition of TNFR1 signaling is a highly sought after strategy for treatment of inflammatory diseases such as rheumatoid arthritis. In this study, we investigated the structure-function relationship of TNFR1 by engineering a TNFR1 fluorescence resonance energy transfer (FRET) biosensor to monitor the structural and conformational changes of the receptor. We have also shown using small-molecule tool compounds, that the disruption of receptor-receptor interactions (competitive inhibition) and perturbation of the receptor conformational dynamics (allosteric inhibition) are both feasible approaches to inhibit TNFR1 signaling. We have also made a major discovery showing that long-range structural couplings, between TNFR1 membrane distal and proximal domains, mediated through the ligand-binding loop, determine the conformational states of the receptor that act as a molecular switch in receptor function. In addition to deepening the understanding of a novel mechanism in TNF receptor activation, we have optimized a lead compound through medicinal chemistry by improving its potency by more than sixty-fold to the nanomolar range, thereby advancing therapeutic developments in these clinically important targets. The heterogeneity of tau and HTT pathology is one of the major challenges that plagues current clinical trials, hence impeding the discovery of a cure for Alzheimer’s disease (AD) and Huntington’s disease (HD). We have engineered novel FRET biosensors of these proteins to target the ensemble of heterogeneous protein oligomers or aggregates in cells. The biosensors are not only capable of monitoring oligomer conformations, but can also be used as a high-throughput screening platform. Using these technologies, we have discovered small-molecule inhibitors of tau oligomerization or HTT aggregation that rescue cell cytotoxicity with nanomolar potency.
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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 Based Approaches to Study Cam-Ryr Structural Interaction
    (2020-07) McCarthy, Megan
    Excitation-contraction coupling in muscle is the physiological process of converting an electrical stimulus to a mechanical response. Release of Ca2+ from intracellular stores is essential for this process and is facilitated by calcium release from ryanodine receptor (RyR) calcium release channels. RyR channels are regulated by numerous small molecules and endogenous proteins, including calmodulin (CaM). CaM is a highly conserved, ubiquitously expressed, small dumb-bell shaped protein that binds four Ca2+ ions via four EF-hand motifs and regulates RyR in a calcium-dependent manner. At low (nM) [Ca2+] CaM is a partial agonist of RyR and it is an inhibitor at high (mM) [Ca2+]. The functional effects of CaM regulation of RyR are well established, but the structural mechanism of Ca2+-dependent regulation of RyR by CaM remains poorly understood. In part, this is due to the large size of RyR (2.2 MDa), which has limited most studies to peptide fragments of the CaM binding domain. The goal of this thesis is to elucidate the Ca2+-dependent conformational changes in CaM when in complex with full-length RyR, to gain insight into the mechanism of CaM-mediated regulation of RyR. Complementary fluorescence resonance energy transfer (FRET) and fluorescence-based stopped-flow kinetics experiments were performed to determine the Ca2+ -dependent structural changes in CaM when in complex with RyR. CaM was labeled with fluorescent probes in each lobe (N- and C-) and time-resolved FRET (TR-FRET) was used to assess inter-lobe distances (Chapter 3). With CaM bound to full-length RyR1, TR-FRET resolved two conformations, and Ca2+ stabilized a closed conformation by a factor of two. Surprisingly, an open conformation was the major component at high and low Ca2+, while the closed conformation was the major component in the presence of a peptide from the CaM binding domain of RyR 1. Calcium cycling in muscle contraction is a fast process, so to understand how Ca2+ binding events mediate conformational change in CaM when bound to full-length RyR1, CaM was labeled with an environment-sensitive probe and the rate of structural transition was monitored by stopped-flow kinetics (Chapter 4). We found differences in the rates of structural transition induced by Ca2+ binding between CaM bound to full-length RyR and a peptide from the CaM binding domain of RyR. These results provide new insights into the structural basis of CaM regulation of RyR1. CaM binds Ca2+ and undergoes structural transitions differently when bound to full-length RyR1 compared to the peptide. These differences may apply to other CaM targets and should motivate more structural work with CaM in the presence of full-length binding partners.
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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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    Myosin structural dynamics: mechanistic insights and therapeutic technology developments
    (2019-03) Rohde, John
    A major focus in molecular biophysics is to understand how protein structural isomerizations correspond to cellular and organismal physiology. The heart generates force to perfuse the body with oxygenated blood through contractile units in myocytes called sarcomeres. The primary force-generating protein in this contractile apparatus is myosin. Our lab has developed a strategic tool called transient time-resolved FRET, (TR)2FRET, to measure directly, with sub-nanometer and sub-millisecond resolution, the structural and biochemical kinetics of muscle myosin. This tool allows us to directly determine how myosin’s power stroke is coupled to the thermodynamic drive for force generation—the entropically-favored dissociation of inorganic phosphate. My research revealed that actin initiates the force-generating power stroke before phosphate dissociation, revealing how power output and efficiency are regulated by the distribution of myosin’s structural states. (TR)2FRET is also a powerful tool to examine small-molecule perturbations of structural transitions within myosin’s kinetic cycle. Omecamtiv mecarbil (OM), a putative heart failure therapeutic, increases cardiac contractility. My results demonstrate that OM stabilizes myosin’s pre-powerstroke structural state and significantly slows the actin-induced powerstroke. I also used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and prospective therapeutic for hypertrophic cardiomyopathy. I found that mavacamten stabilizes an auto-inhibited state of two-headed cardiac myosin, not present in the single-headed myosin motor fragment. From these results, we predicted that cardiac myosin is regulated by an interaction between its two heads and the thick filament, and proposed that mavacamten stabilizes this state. I also investigated two mutations in the converter domain of myosin V to examine how point mutations alter specific structural transitions in the myosin motor’s ATPase cycle. Transient kinetics analyses and FRET-based experiments demonstrated that one mutation slowed the recovery-stroke rate constants, while a second mutation enhanced these steps. These mutations correspond to human mutations that give rise to dilated or hypertrophic cardiomyopathies, respectively. Together these experiments reveal new and important mechanistic insights into myosin’s structural dynamics and provide proof-of-concept results for developing therapeutic technology.
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    Novel Fluorescence Tools for the Discovery of Cardiac Calcium Pump Therapeutics
    (2017-02) Schaaf, Tory
    The sarco/endoplasmic reticulum calcium ATPase (SERCA) is the calcium pump responsible for maintaining cellular calcium homeostasis. Diminished SERCA function has been directly linked to numerous degenerative disease states, such as heart failure. The pathological progression of heart failure is associated with an elevated level of cytosolic calcium, and impairs the function of the muscle contraction-cycle. The overarching goal of this research is to discover novel small-molecule effectors, capable of enhancing SERCA’s ability to pump and store calcium within the sarcoplasmic reticulum (SR). Drugs that increase the calcium pumping efficiency of SERCA will restore calcium homeostasis by reducing the calcium content in the cytosol, and enhance impaired cardiac function. The process of drug discovery is a high-risk effort, and involves screening millions of small-molecules to fortuitously discover a lead compound with high-therapeutic potential. The precise placement of two fluorescent proteins at specific locations along SERCA’s cytosolic headpiece, allows for the detection of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorescent proteins. Human cell lines that overexpress this fluorescent fusion protein were generated, creating a live-cell biosensor. The rate of energy transfer (FRET) is dependent on the distance between the fluorescent probes and linked to the enzymatic activity of SERCA. FRET tracks SERCA’s structural status, while it pumps calcium into the sarcoplasmic reticulum. These biosensors are grown in vast quantities, harvested, and utilized for high-throughput drug screening. The cells are dispensed into high-density microplates, where each well contains a different compound. FRET is detected using proprietary fluorescence technology, capable of recording the nanosecond fluorescence decay rate (lifetime) and the full emission spectrum. Both lifetime and spectral modes offer incredibly fast speeds, with high resolution and precision. High-throughput screening by lifetime mode offers the advantage of resolving the structural status of the FRET biosensor because the mole fraction of each structural state is assessed, and candidate compounds found during the screening process can be characterized by their structural effect on the biosensor. High-throughput screening by spectral mode increases assays precision by taking into account the shape of the fluorescence emission spectrum. The shapes of these spectra are decomposed into the contribution of known components by a novel spectral unmixing method, and further used to accurately evaluate FRET. When coupled with lifetime mode, spectral-based drug screening increases assay precision and removes artifacts from cellular autofluorescence and fluorescent compounds. The complementary advantages of coupling spectral and lifetime fluorescence measurements significantly reduces the rate of false-positives from high-throughput drug screens. The development of the technology and FRET biosensor assay, drastically increases the probability of identifying a novel drug with great therapeutic potential.
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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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    The Role of Cardiac Troponin I as an Allosteric Regulator of Sarcomere Activation
    (2017-12) Vetter, Anthony
    The cardiac sarcomere is the functional unit of force production in the heart. The sarcomere is a highly ordered near-liquid crystalline arrays of thin and thick filament proteins held in stoichiometric balance that work in a concerted fashion to orchestrate the heart’s pump function. Regulation of cardiac output is mediated by a combination of myocyte cell-intrinsic and cell-extrinsic factors that fine-tune the contractile machinery. Central to the regulation of the sarcomere is the heterotrimeric cardiac troponin complex (cTn) whose subunits include: cardiac troponin T (cTnT), an adaptor protein that tethers cTn into the ultrastructure of the thin filament via tropomyosin; cardiac troponin C (cTnC), a calmodulin-like EF-hand protein that confers calcium sensitivity to the thin filament; and cardiac troponin I (cTnI), a molecular switch that toggles between the actin filament and N-terminal lobe of cTnC. When associated with actin, cTnI prevents the azimuthal rotation of tropomyosin along the filament thereby concealing strong myosin binding sites along the thin filament. Therefore, cTn may be thought of as a binary switch regulated by calcium, toggling cTnI between the actin-associated inhibitory state and the cTnC-associated active state. The penultimate outcome of this dynamic structural process is changing the steric accessibility of myosin binding sites to cycling force producing myosin cross-bridges. Alterations in the sarcomeric structure-function relationship by both cell-intrinsic and cell-extrinsic factors underlie the pathology of numerous acquired and inherited cardiomyopathies. As such, gaining a greater understanding of the activation, inactivation, and molecular interactions within the sarcomere underpins and enables our ability to redress heart failure. For decades, tools have long been available to quantitatively detect and monitor the sarcolemmal depolarization and calcium transient that initiate contraction. Furthermore, the final output of the contractile machinery can be monitored by a variety of tools that measure the force production of the sarcomere. However, to this date, there has been a “black box” around the intervening processes which govern the sarcomere activation under physiological conditions. A preponderance of evidence derived from studies of isolated proteins, electron microscopy, and permeabilized steady-state muscle fibers has supported the theory that strong myosin binding is requisite to activate the sarcomere. However informative these studies are, they are limited by their non-physiological experimental conditions. Here, ex vivo cellular physiology enabled by a novel live-cell biosensor and in silico molecular dynamics simulations were used in tandem to investigate the activation of the sarcomere and specifically how the cTnC:cTnI interface is a critical juncture for determining contractility under physiological conditions. Evidence suggests that in juxtaposition to the long-standing three-state model wherein myosin binding to actin is necessary to shift the equilibrium of cTn complexes from the closed to the open state, calcium and cTnI switch peptide binding are sufficient to activate the sarcomere and permit force production by cycling cross-bridges under the live cell conditions described herein. Furthermore, when cTnI binds to the N-terminal lobe of cTnC, the interaction between helix 4 of cTnI and the A helix of cTnC is a primary determinant of contractility independent of changes in the calcium-transient. Collectively, this work enhances our knowledge of how the cardiac sarcomere is regulated and establishes it as a potent therapeutic target to improve heart pump function in ailing myocardium.
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    Rotational dynamics-based assessment of energy transfer efficiency of hetero-FRET probes in crowded environments
    (2017-08) Leopold, Hannah
    A living cell is crowded with various organelles, DNA, and proteins. Such macromolecular crowding has a significant impact on cellular processes. Yet, the effects of macromolecular crowding on protein diffusion, reaction rates, and folding are far from understood. As a result, there is a need to quantify crowding in a heterogeneous environment both in vivo and in vitro. Recently, a series of novel genetically encoded FRET probes were developed as sensors to quantitatively measure crowding in vivo and were characterized with steady-state fluorescence (Nat Meth [2015] 12:227). In a crowded environment, these FRET probes are hypothesized to become confined and more compact, thereby leading to enhanced energy transfer. Consequently, the level of crowding can be quantified based on the energy transfer efficiency of the probes. In this Thesis, we develop a theoretical model based on time-resolved anisotropy to quantify the FRET efficiency of the probes. Additionally, we investigate the conformational dynamics and rotational diffusion of the probes using time-resolved fluorescence anisotropy in homogeneous and heterogeneous environments. Here, we used Ficoll-70 as a heterogeneous crowder to investigate the excluded volume effects on the probes. Measurements in glycerol-enriched buffer were also conducted to distinguish between viscosity and excluded volume effects. Our results indicate that time-resolved anisotropy can be combined with these novel FRET probes for quantitative, non-invasive analysis of site-specific crowding.
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    Single-molecule studies of novel, genetically encoded, hetero-FRET sensors to environmental ionic-strength using different modalities of fluorescence correlation spectroscopy (FCS)
    (2020-08) Kay, Taryn
    A living cell is a complex environment with heterogeneous and dynamic distributions of ionic strength and macromolecular crowding. Ionic strength influences many aspects of the biology of living cells such as catalytic activities of enzymes, cell volume, osmosis, and protein functions. The challenge, however, is that the ionic strength varies, both spatially and temporarily, throughout the milieu of living cells. As a result, there is a need for ionic-strength sensors that can be genetically encoded in different compartments in living cells, while being amenable to quantitative and noninvasive analytical methods. Importantly, low-level expression of those potential ionic-strength sensors is desirable such that they will not interfere with the function and biological activities of the native protein. In this project, we investigate a family of genetically encoded ionic-strength sensors (mCerulean3-linker-mCitrine) that consist of a donor (mCerulean3), an acceptor (mCitrine), and a linker region made of two oppositely charged α-helices at the single-molecule level. We hypothesize that as ionic strength in the environment increases, the electrostatic attraction between the charged helices will decrease, pulling the donor and acceptor apart, and therefore decreasing energy transfer efficiency at the single-molecule level. To test this hypothesis, we have developed a new approach based on the molecular brightness of the cleaved and intact sensors (RD and KE) for FRET analysis using fluorescence correlation spectroscopy (FCS) in 10 mM sodium phosphate buffer as a function of the environmental ionic strength using different salts. Towards these goals, we rebuilt, calibrated, and optimized a home-built FCS setup, which was used to laser wavelength dependent studies of the fluorescence fluctuation autocorrelation analysis of these sensors. In addition, we characterized the translational diffusion coefficient and hydrodynamic radius of these sensors under different laser wavelengths and compared our results using theoretical model that relate the hydrodynamic volume with the molecular weight of proteins. Our single-molecule approach for FRET analysis of genetically encoded donor-acceptor pairs are particularly amenable to live cell studies with the added advantage of requiring very low expression levels of the sensor as compared with conventional, ensemble-based methods.
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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 dynamics of the myosin force-generating region.
    (2009-12) Agafonov, Roman
    Myosin is a molecular motor that generates force on actin using energy from ATP hydrolysis. Myosin plays a key role in muscle contraction and is responsible for a variety of motility processes at the cellular level. It works cyclically, changing its conformation during the power stroke and the recovery stroke. X-ray crystallography has provided information about the structural organization of myosin in different biochemical states (as defined by bound nucleotide), inspiring several structural models that could explain the molecular mechanism of myosin's function. Spectroscopy, in combination with site-directed labeling and transient experiments, can test and refine these models and provide information about myosin's dynamic properties. The goal of this project was to determine the structural dynamics of the myosin force-generating domain and study coupling mechanisms between this domain and the myosin active site. We have chosen Dictyostelium discoideum (Dicty) as our experimental system since it provides multiple advantages in comparison with the muscle myosin. In particular, it is possible to manipulate the Dicty DNA sequence, engineering labeling sites at desired locations and introducing functional mutations at the points of interest. As a first part of the project, we have tested Dicty myosin in comparison with myosin purified from rabbit skeletal muscle, and have shown that structural changes in the force-generating domain of Dicty and rabbit myosin are identical. We then focused on specific elements within the force-generating domain, relay helix and relay loop, as these elements appeared to be crucial for interdomain coupling and force generation. Using time-resolved EPR and FRET, we have developed a spectroscopic approach to determine the conformation of the relay helix. We have also developed a novel technique that we called transient time-resolved FRET [(TR)2FRET], which allowed us to monitor structural changes within the relay helix in real time. We then studied the relationship between the state of the myosin active site (which is determined by the bound ligand) and the structure of the relay helix. To obtain insights about regulatory mechanisms, we have investigated the effect of a mutation that is known to abolish myosin motor function, despite leaving enzymatic activity intact. These experiments revealed important coupling mechanisms between the relay loop and relay helix, providing a structural explanation for the previously observed functional effects and a model for power stroke activation in myosin.
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    Targeting Pathological Protein Interactions in Drug Discovery
    (2021-07) Young, Malaney
    The interaction of proteins with themselves or other molecules is essential to biological function. The dysregulation of normal protein structure, folding, and interactions forms the basis of many human diseases. Elucidating the protein interactions which underlie various disease states is crucial to understanding disease progression and identifying protein targets for therapeutic development. This dissertation focuses on targeting pathological protein interactions in cancer, non-alcoholic fatty liver disease (NAFLD), and neurodegenerative disorders. The tools developed in this dissertation are not only relevant to these specific disease states, but can be modified to pursue novel protein targets for drug discovery in other diseases as well. Chapter 1 of this dissertation provides an explanation of the scientific background and previous research that has motivated the current work. Chapter 2 presents the first high-throughput screening (HTS) platform that detects structural changes in death receptor 5 (DR5), a membrane protein which regulates apoptosis. In this chapter we have identified small-molecules which modulate DR5 signaling and sensitize TRAIL-resistant cancer cells to TRAIL-induced apoptosis. In chapter 3, the same HTS platform is used to discover novel inhibitors of DR5-mediated TRAIL-induced apoptosis for therapeutic intervention of NAFLD and Alzheimer’s Disease. Chapter 4 provides a summary of various projects which involved research on alpha-synuclein (α-syn), the primary protein involved in Parkinson’s Disease. In chapter 4.2, yeast display is implemented as a screening platform to identify inhibitors of α-syn uptake via targeting neurexin-1β. Chapter 4.3 provides an in-depth protocol on the purification of α-syn for laboratory studies. Chapter 4.4 summarizes an attempt to create a cell-free FRET-based biosensor to target the pathological aggregation of α-syn. Chapter 4.5 details a protocol for producing dopamine-modified α-syn oligomers for laboratory studies on dopamine-induced α-syn toxicity. Finally, chapter 4.6 identifies threonine 75 in α-syn as a critical amino acid for fibrillization. This work has overall contributed to the field of drug discovery by providing novel high-throughput screening tools to study DR5 and other protein-protein interactions, and has further elucidated important aspects of α-syn aggregation to be targeted in Parkinson’s Disease.