title: Characterization of Zebrafish Cardiac and Slow Skeletal Troponin C Paralogs by MD Simulation and ITC.
abstract: Zebrafish, as a model for teleost fish, have two paralogous troponin C (TnC) genes that are expressed in the heart differentially in response to temperature acclimation. Upon Ca(2+) binding, TnC changes conformation and exposes a hydrophobic patch that interacts with troponin I and initiates cardiac muscle contraction. Teleost-specific TnC paralogs have not yet been functionally characterized. In this study we have modeled the structures of the paralogs using molecular dynamics simulations at 18°C and 28°C and calculated the different Ca(2+)-binding properties between the teleost cardiac (cTnC or TnC1a) and slow-skeletal (ssTnC or TnC1b) paralogs through potential-of-mean-force calculations. These values are compared with thermodynamic binding properties obtained through isothermal titration calorimetry (ITC). The modeled structures of each of the paralogs are similar at each temperature, with the exception of helix C, which flanks the Ca(2+) binding site; this region is also home to paralog-specific sequence substitutions that we predict have an influence on protein function. The short timescale of the potential-of-mean-force calculation precludes the inclusion of the conformational change on the ΔG of Ca(2+) interaction, whereas the ITC analysis includes the Ca(2+) binding and conformational change of the TnC molecule. ITC analysis has revealed that ssTnC has higher Ca(2+) affinity than cTnC for Ca(2+) overall, whereas each of the paralogs has increased affinity at 28°C compared to 18°C. Microsecond-timescale simulations have calculated that the cTnC paralog transitions from the closed to the open state more readily than the ssTnC paralog, an unfavorable transition that would decrease the ITC-derived Ca(2+) affinity while simultaneously increasing the Ca(2+) sensitivity of the myofilament. We propose that the preferential expression of cTnC at lower temperatures increases myofilament Ca(2+) sensitivity by this mechanism, despite the lower Ca(2+) affinity that we have measured by ITC.
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada; Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Stevens
authors.affiliation: Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada.
authors.forename: Kaveh
authors.initials: K
authors.lastname: Rayani
authors.affiliation: Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Genge
authors.affiliation: Biocomputing Group, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Gurpreet
authors.initials: G
authors.lastname: Singh
authors.affiliation: Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada.
authors.forename: Bo
authors.initials: B
authors.lastname: Liang
authors.affiliation: Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada.
authors.forename: Janine M
authors.initials: JM
authors.lastname: Roller
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
authors.forename: Cindy
authors.initials: C
authors.lastname: Li
authors.affiliation: Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada.
authors.forename: Alison Yueh
authors.initials: AY
authors.lastname: Li
authors.affiliation: Biocomputing Group, University of Calgary, Calgary, Alberta, Canada.
authors.forename: D Peter
authors.initials: DP
authors.lastname: Tieleman
authors.affiliation: Department of Biochemistry, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Filip
authors.initials: F
authors.lastname: van Petegem
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada; Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada; Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada. Electronic address: tibbits@sfu.ca.
authors.forename: Glen F
authors.initials: GF
authors.lastname: Tibbits
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002151:Calorimetry; D056004:Molecular Dynamics Simulation; D018482:Muscle, Skeletal / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D019209:Troponin C / Q000737:chemistry* / Q000378:metabolism*; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D019209:Troponin C; D029961:Zebrafish Proteins; D002118:Calcium