Browsing by Subject "calmodulin"

Now showing 1 - 3 of 3
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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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    Positive And Negative Regulation Of Defense Responses Against Pseudomonas Syringae In Arabidopsis
    (2014-03) Sreekanta, Suma
    Immune signaling in plants involves both positive and negative regulators. Maintaining a balance between growth and defense responses is important because there is a fitness cost to the plants if immune responses are left unchecked. Suppression of immune responses in the absence of pathogens as well as after the threat has passed is critical in maintaining such a balance between growth and defense responses. Upon pathogen perception, the positive regulators counter the immune repression to induce defense responses. We investigated the roles of two genes, CBP60a and PCRK1 in the regulation of defense responses against Pseudomonas syringae pathogen in the model system Arabidopsis thaliana . CBP60a is a negative regulator of immune responses. We showed that CBP60a is a CaM binding protein and that CaM binding is important for its function in transducing defense signals. Mutants of CBP60a were more resistant to Pseudomonas syringae infection suggesting that CBP60a was a negative regulator of defense responses. We found that CBP60a functions in repressing immune signaling under conditions where the plants are not challenged by a pathogen. We also investigated the role of a putative kinase, PCRK1, in immune signaling. We showed that pcrk1 mutants are more susceptible to Pseudomonas syringae than wild type plants suggesting that PCRK1 has a positive role in immune responses. We also showed that PCRK1 is important for immunity triggered by some of the conserved Microbe Associated Molecular Patterns (MAMP) as well endogenous signals generated as a result of pathogen activity called Damage Associated Molecular Patterns (DAMP).
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    Structural Dynamics of the Calmodulin-Ryanodine Receptor Interaction Using Bifunctional Spin Labels and EPR
    (2018-08) Her, Cheng
    Muscle contraction and relaxation are regulated by changes in intracellular calcium levels. To facilitate muscle contraction, calcium is released from the intracellular calcium reservoir into the cytosol by the homotetrameric calcium channel known as the ryanodine receptor (RyR). The sarcoplasmic reticulum membrane-embedded RyR is a target for many small molecule and protein modulators, including the ubiquitously expressed calcium binding protein calmodulin (CaM). CaM can bind four calcium ions via its four EF-hand motifs and has calcium-dependent effects on RyR. It is well established that CaM potentiates channel opening below µM calcium and inhibition above µM calcium. Despite this, the structural mechanism of the calcium-dependent CaM-mediated RyR regulation remain poorly understood. The primary goal of the work presented here is to elucidate the structural mechanisms of the CaM-RyR interaction, using bifunctional spin labels and electron paramagnetic resonance (EPR). In the first study, we investigated the structural dynamics of a spin labeled ryanodine receptor peptide (RyRp) bound to CaM using EPR (Chapter 4). By detecting the rotational dynamics of specific sites along the backbone, we show that the interaction of RyRp with CaM is nonuniform along the peptide, and the primary effect of calcium is to increase the interaction of the N-lobe of CaM with RyRp. In the second study (Chapter 5), we placed spin probes on both CaM and RyRp and investigated the calciumdependent structural changes of the complex using a distance measurement EPR technique known as double electron-electron resonance (DEER). Our DEER distance results provide support for the conformational selection mechanism of CaM binding to RyRp (i.e. the binding of RyRp shifts CaM to preexisting structural states). We discovered differential Ca effects on the two lobes of CaM with respect to RyRp binding. More specifically, we discovered that Ca was required for complete interaction of the N-lobe with RyRp, while the C-lobe bound RyRp independent of Ca. These findings are consistent with results from Chapter 4 and provide support for the hypothesis that CaM functions as a subunit of RyR through binding of the C-lobe, and complete interaction of the N-lobe of CaM (in response to increased cytosolic Ca levels) is responsible for maximum inhibition of RyR. Thus, our results provide novel insight into the structural mechanism of CaM-mediated RyR regulation while showcasing an innovative approach with wide applicability to other biological systems.