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Fluorescent Protein-Based Ca2+ Sensor Reveals Global, Divalent Cation-Dependent Conformational Changes in Cardiac Troponin C
| Title: | Fluorescent Protein-Based Ca2+ Sensor Reveals Global, Divalent Cation-Dependent Conformational Changes in Cardiac Troponin C. |
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| Name(s): |
Badr, Myriam A., author Pinto, Jose R., author Davidson, Michael W., author Chase, P. Bryant, author |
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| Type of Resource: | text | |
| Genre: | Text | |
| Date Issued: | 2016-10-13 | |
| Physical Form: |
computer online resource |
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| Extent: | 1 online resource | |
| Language(s): | English | |
| Abstract/Description: | 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 similar to 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. | |
| Identifier: | FSU_libsubv1_wos_000385505800046 (IID), 10.1371/journal.pone.0164222 (DOI), PMC5063504 (PMCID) | |
| Keywords: | calcium-binding, genetically encoded indicators, hypertrophic cardiomyopathy, magnesium binding-sites, mutations, myofibrillar adenosine-triphosphatase, neutron-scattering, regulatory domain, skeletal-muscle fibers, thin-filament | |
| Publication Note: | The publisher’s version of record is available at http://www.dx.doi.org/10.1371/journal.pone.0164222 | |
| Persistent Link to This Record: | http://purl.flvc.org/fsu/fd/FSU_libsubv1_wos_000385505800046 | |
| Host Institution: | FSU | |
| Is Part Of: |
Plos One. 1932-6203 |
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| Issue: | iss. 10, vol. 11 |
Badr, M. A., Pinto, J. R., Davidson, M. W., & Chase, P. B. (2016). Fluorescent Protein-Based Ca2+ Sensor Reveals Global, Divalent Cation-Dependent Conformational Changes in Cardiac Troponin C. Plos One. Retrieved from http://purl.flvc.org/fsu/fd/FSU_libsubv1_wos_000385505800046
