title: Fluorescent Protein-Based Ca2+ Sensor Reveals Global, Divalent Cation-Dependent Conformational Changes in Cardiac Troponin C.
abstract: Cardiac troponin C (cTnC) is a key effector in cardiac muscle excitation-contraction coupling as the Ca2+ sensing subunit responsible for controlling contraction. In this study, we generated several FRET sensors for divalent cations based on cTnC flanked by a donor fluorescent protein (CFP) and an acceptor fluorescent protein (YFP). The sensors report Ca2+ and Mg2+ binding, and relay global structural information about the structural relationship between cTnC's N- and C-domains. The sensors were first characterized using end point titrations to decipher the response to Ca2+ binding in the presence or absence of Mg2+. The sensor that exhibited the largest responses in end point titrations, CTV-TnC, (Cerulean, TnC, and Venus) was characterized more extensively. Most of the divalent cation-dependent FRET signal originates from the high affinity C-terminal EF hands. CTV-TnC reconstitutes into skinned fiber preparations indicating proper assembly of troponin complex, with only ~0.2 pCa unit rightward shift of Ca2+-sensitive force development compared to WT-cTnC. Affinity of CTV-TnC for divalent cations is in agreement with known values for WT-cTnC. Analytical ultracentrifugation indicates that CTV-TnC undergoes compaction as divalent cations bind. C-terminal sites induce ion-specific (Ca2+ versus Mg2+) conformational changes in cTnC. Our data also provide support for the presence of additional, non-EF-hand sites on cTnC for Mg2+ binding. In conclusion, we successfully generated a novel FRET-Ca2+ sensor based on full length cTnC with a variety of cellular applications. Our sensor reveals global structural information about cTnC upon divalent cation binding.
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, United States of America.|Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, United States of America.
authors.forename: Myriam A
authors.initials: MA
authors.lastname: Badr
authors.affiliation: Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida, United States of America.
authors.forename: Jose R
authors.initials: JR
authors.lastname: Pinto
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, United States of America.|National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, United States of America.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Davidson
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, United States of America.|Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, United States of America.
authors.forename: P Bryant
authors.initials: PB
authors.lastname: Chase
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism*; D008274:Magnesium; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D019209:Troponin C / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D008164:Luminescent Proteins; D019209:Troponin C; D008274:Magnesium; D002118:Calcium