- Generation and Characterization of Fret Constructs of Cardiac Troponin C to Study Divalent Cation-Dependent Structural Changes and Develop a Ca2+ Sensor.
Badr, Myriam Antoine, Chase, P. Bryant, Overton, Michael, Fajer, Piotr, Keller, Thomas C. S., Strouse, Geoffrey, Program in Molecular Biophysics, Florida State University
Troponin C (TnC), the Ca2+ binding subunit in striated muscle, is central to regulation of contraction. Binding of Ca2+ to TnC results in a series of conformational changes in the different regulatory proteins, which ultimately leads to muscle contraction. From structural studies about Troponin C, we hypothesize that binding of divalent cations causes a closing in the molecule, bringing the ends of TnC closer together. We designed a series of novel FRET constructs using human cTnC, to examine...
Show moreTroponin C (TnC), the Ca2+ binding subunit in striated muscle, is central to regulation of contraction. Binding of Ca2+ to TnC results in a series of conformational changes in the different regulatory proteins, which ultimately leads to muscle contraction. From structural studies about Troponin C, we hypothesize that binding of divalent cations causes a closing in the molecule, bringing the ends of TnC closer together. We designed a series of novel FRET constructs using human cTnC, to examine the relative positions of the N- and C-termini upon divalent cation binding. Full length cTnC was flanked by FRET pairs of fluorescent proteins (mCerulean/mVenus, mTurquoise/mNeonGreen, mTurquoise/cpVenus), varying the linker length between TnC and the FRET proteins. FRET, as quantified by changes in the fluorescence ratio (FR) of acceptor to donor, was measured in the presence and absence of saturating Ca2+ and/or Mg2+. FRET increased substantially and reversibly upon Ca2+ binding to cTnC. Similar FRET changes were observed upon saturation of the C-terminus with Mg2+, suggesting that the structural changes detected are primarily attributable to occupancy of the C-terminal sites. We chose to further characterize our most successful construct CTV-TnC by measuring conformational changes by AUC. We also confirmed the functionality of our construct with skinned fiber force measurements. In addition, we have conducted FRET and AUC experiment with mutant TnC where one or more EF hand was inactivated, to extract the contribution of each EF hand to the total signal. Analytical ultracentrifugation (AUC) confirmed that constructs undergo global conformational changes to a more compact structure upon Mg2+ binding, with further compaction when Ca2+ occupies all 3 sites of cTnC. Fiber experiments have shown that CTV-TnC is capable of reconstituting into the sarcomere and generating force under activating conditions, with a slight decrease in Ca2+ sensitivity. Finally, we fully titrated CTV-TnC with Ca2+ and Mg2+ to determine affinities of the divalent cation to the EF hands, using the FR signal. The full divalent cation titration generally agrees with known affinities of WT TnC. Finally, studies of the mutant TnC show that the N-terminus is associated with a negative FRET signal, which requires more experiments to be fully understood. The C-terminal sites show a difference in contribution when bound to Ca2+ vs. Mg2+, with the highest contribution of site III upon Ca2+ binding, and the highest contribution of site IV upon Mg2+ binding. In summary, we have successfully designed a Ca2+ sensor capable of indicating Ca2+ binding through large changes in FRET signal. This sensor is capable of reconstituting into troponin complex, as well as into sarcomeres, making it useful to the muscle research community. The FRET and AUC studies show that the ends of cTnC come closer upon binding divalent cations yielding a more compact structure, for CTV-TnC alone and within the troponin complex. Our study also indicates a difference in site III and IV, known as the C-terminal sites, in the contribution to the FRET signal when bound to each of the divalent cations Ca2+ or Mg2+.
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Current Search: Thomas, Geoffrey P.