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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
Type of Resource: text
Genre: Journal Article
Text
Date Issued: 2016-10-13
Physical Form: computer
online resource
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 ~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_pmch_27736894 (IID), 10.1371/journal.pone.0164222 (DOI), PMC5063504 (PMCID), 27736894 (RID), 27736894 (EID), PONE-D-16-17782 (PII)
Grant Number: R00 HL103840, R01 HL128683
Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063504.
Subject(s): Biosensing Techniques/instrumentation
Calcium/metabolism
Cations, Divalent/chemistry
Cations, Divalent/metabolism
Crystallography, X-Ray
Humans
Luminescent Proteins/chemistry
Luminescent Proteins/metabolism
Magnesium
Models, Molecular
Protein Binding
Protein Structure, Secondary
Troponin C/chemistry
Troponin C/metabolism
Persistent Link to This Record: http://purl.flvc.org/fsu/fd/FSU_pmch_27736894
Owner Institution: FSU
Is Part Of: PloS one.
1932-6203
Issue: iss. 10, vol. 11

Choose the citation style.
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_pmch_27736894