The Experts below are selected from a list of 201 Experts worldwide ranked by ideXlab platform

David D. Thomas - One of the best experts on this subject based on the ideXlab platform.

  • Probing the Molecular Mechanism of SERCA-PLB Regulation by Time-Resolved FRET
    Biophysical Journal, 2011
    Co-Authors: Elizabeth L. Lockamy, Razvan L. Cornea, Christine B. Karim, David D. Thomas
    Abstract:

    We are using solid-phase peptide synthesis, membrane reconstitution, an enzyme-coupled Ca-ATPase Activity Assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA Activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA Activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase Activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA.

  • Probing the Mechanism of SERCA-PLB Regulation by Time-Resolved Fret
    Biophysical Journal, 2010
    Co-Authors: Elizabeth L. Lockamy, Razvan L. Cornea, Christine B. Karim, David D. Thomas
    Abstract:

    We are using solid-phase peptide synthesis, membrane reconstitution, an enzyme-coupled Ca-ATPase Activity Assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA Activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA Activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase Activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight but opposite effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA, with distinct mechanisms for relief by Ca2+ and PLB phosphorylation.

Elizabeth L. Lockamy - One of the best experts on this subject based on the ideXlab platform.

  • Probing the Molecular Mechanism of SERCA-PLB Regulation by Time-Resolved FRET
    Biophysical Journal, 2011
    Co-Authors: Elizabeth L. Lockamy, Razvan L. Cornea, Christine B. Karim, David D. Thomas
    Abstract:

    We are using solid-phase peptide synthesis, membrane reconstitution, an enzyme-coupled Ca-ATPase Activity Assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA Activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA Activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase Activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA.

  • Probing the Mechanism of SERCA-PLB Regulation by Time-Resolved Fret
    Biophysical Journal, 2010
    Co-Authors: Elizabeth L. Lockamy, Razvan L. Cornea, Christine B. Karim, David D. Thomas
    Abstract:

    We are using solid-phase peptide synthesis, membrane reconstitution, an enzyme-coupled Ca-ATPase Activity Assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA Activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA Activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase Activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight but opposite effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA, with distinct mechanisms for relief by Ca2+ and PLB phosphorylation.

John E. Scott - One of the best experts on this subject based on the ideXlab platform.

  • development and validation of a high throughput intrinsic ATPase Activity Assay for the discovery of mekk2 inhibitors
    Journal of Biomolecular Screening, 2013
    Co-Authors: Syed Ahmad, Mark A. Hughes, Gary L. Johnson, John E. Scott
    Abstract:

    The kinase MEKK2 (MAP3K2) has recently been implicated in tumor growth and metastasis. Thus, selective inhibition of MEKK2 may be a novel strategy for cancer therapy. To identify inhibitors of MEKK2 kinase Activity, we have developed a novel Activity Assay for MEKK2 based on the discovery that recombinant purified MEKK2 has intrinsic ATPase Activity. This MEKK2 ATPase Assay was validated for enzyme identity and enzymatic purity by multiple methods including mass spectrometry analysis, testing different sources of MEKK2 and comparing ATPase Assay IC 50 data for multiple inhibitors to literature values and to IC 50 data generated using MEKK2 binding and transphosphorylation Assays. Taken together, these data indicated that genuine MEKK2 Activity was being measured in this Assay and no other ATPases contributed to the signal. A miniaturized version of the Assay was validated for high-throughput screening, and compound libraries were screened. The screening hits generated comparable potencies in the MEKK2 intrinsic ATPase, binding, and transphosphorylation Assays. We identified a novel MEKK2 inhibitor and confirmed that crizotinib and bosutinib are potent in vitro inhibitors of MEKK2 Activity with IC 50 values of <100 nM. Thus, this Assay has utility for the discovery of small-molecule inhibitors of MEKK2 Activity.

  • Development and validation of a high-throughput intrinsic ATPase Activity Assay for the discovery of MEKK2 inhibitors.
    Journal of biomolecular screening, 2012
    Co-Authors: Syed Ahmad, Mark A. Hughes, Gary L. Johnson, John E. Scott
    Abstract:

    The kinase MEKK2 (MAP3K2) has recently been implicated in tumor growth and metastasis. Thus, selective inhibition of MEKK2 may be a novel strategy for cancer therapy. To identify inhibitors of MEKK2 kinase Activity, we have developed a novel Activity Assay for MEKK2 based on the discovery that recombinant purified MEKK2 has intrinsic ATPase Activity. This MEKK2 ATPase Assay was validated for enzyme identity and enzymatic purity by multiple methods including mass spectrometry analysis, testing different sources of MEKK2 and comparing ATPase Assay IC 50 data for multiple inhibitors to literature values and to IC 50 data generated using MEKK2 binding and transphosphorylation Assays. Taken together, these data indicated that genuine MEKK2 Activity was being measured in this Assay and no other ATPases contributed to the signal. A miniaturized version of the Assay was validated for high-throughput screening, and compound libraries were screened. The screening hits generated comparable potencies in the MEKK2 intrinsic ATPase, binding, and transphosphorylation Assays. We identified a novel MEKK2 inhibitor and confirmed that crizotinib and bosutinib are potent in vitro inhibitors of MEKK2 Activity with IC 50 values of

Christine B. Karim - One of the best experts on this subject based on the ideXlab platform.

  • Probing the Molecular Mechanism of SERCA-PLB Regulation by Time-Resolved FRET
    Biophysical Journal, 2011
    Co-Authors: Elizabeth L. Lockamy, Razvan L. Cornea, Christine B. Karim, David D. Thomas
    Abstract:

    We are using solid-phase peptide synthesis, membrane reconstitution, an enzyme-coupled Ca-ATPase Activity Assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA Activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA Activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase Activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA.

  • Probing the Mechanism of SERCA-PLB Regulation by Time-Resolved Fret
    Biophysical Journal, 2010
    Co-Authors: Elizabeth L. Lockamy, Razvan L. Cornea, Christine B. Karim, David D. Thomas
    Abstract:

    We are using solid-phase peptide synthesis, membrane reconstitution, an enzyme-coupled Ca-ATPase Activity Assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA Activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA Activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase Activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight but opposite effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA, with distinct mechanisms for relief by Ca2+ and PLB phosphorylation.

Razvan L. Cornea - One of the best experts on this subject based on the ideXlab platform.

  • Probing the Molecular Mechanism of SERCA-PLB Regulation by Time-Resolved FRET
    Biophysical Journal, 2011
    Co-Authors: Elizabeth L. Lockamy, Razvan L. Cornea, Christine B. Karim, David D. Thomas
    Abstract:

    We are using solid-phase peptide synthesis, membrane reconstitution, an enzyme-coupled Ca-ATPase Activity Assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA Activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA Activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase Activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA.

  • Probing the Mechanism of SERCA-PLB Regulation by Time-Resolved Fret
    Biophysical Journal, 2010
    Co-Authors: Elizabeth L. Lockamy, Razvan L. Cornea, Christine B. Karim, David D. Thomas
    Abstract:

    We are using solid-phase peptide synthesis, membrane reconstitution, an enzyme-coupled Ca-ATPase Activity Assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA Activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA Activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase Activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight but opposite effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA, with distinct mechanisms for relief by Ca2+ and PLB phosphorylation.