Fluorescence resonance energy transfer between residues on troponin and tropomyosin in the reconstituted thin filament: modeling the troponin-tropomyosin complex |
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Authors: | Kimura-Sakiyama Chieko Ueno Yutaka Wakabayashi Katsuzo Miki Masao |
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Institution: | 1 Division of Applied Chemistry and Biotechnology, Graduate School of Engineering Science, Fukui University, Fukui 910-8507, Japan 2 Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8568, Japan 3 Division of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan 4 Research and Education Program for Life Science, Fukui University, Fukui 910-8507, Japan |
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Abstract: | Troponin (Tn), in association with tropomyosin (Tm), plays a central role in the calcium regulation of striated muscle contraction. Fluorescence resonance energy transfer (FRET) between probes attached to the Tn subunits (TnC, TnI, TnT) and to Tm was measured to study the spatial relationship between Tn and Tm on the thin filament. We generated single-cysteine mutants of rabbit skeletal muscle α-Tm, TnI and the β-TnT 25-kDa fragment. The energy donor was attached to a single-cysteine residue at position 60, 73, 127, 159, 200 or 250 on TnT, at 98 on TnC and at 1, 9, 133 or 181 on TnI, while the energy acceptor was located at 13, 146, 160, 174, 190, 209, 230, 271 or 279 on Tm. FRET analysis showed a distinct Ca2+-induced conformational change of the Tm-Tn complex and revealed that TnT60 and TnT73 were closer to Tm13 than Tm279, indicating that the elongated N-terminal region of TnT extends beyond the beginning of the next Tm molecule on the actin filament. Using the atomic coordinates of the crystal structures of Tm and the Tn core domain, we searched for the disposition and orientation of these structures by minimizing the deviations of the calculated FRET efficiencies from the observed FRET efficiencies in order to construct atomic models of the Tn-Tm complex with and without bound Ca2+. In the best-fit models, the Tn core domain is located on residues 160-200 of Tm, with the arrowhead-shaped I-T arm tilting toward the C-terminus of Tm. The angle between the Tm axis and the long axis of TnC is ∼ 75° and ∼ 85° with and without bound Ca2+, respectively. The models indicate that the long axis of TnC is perpendicular to the thin filament without bound Ca2+, and that TnC and the I-T arm tilt toward the filament axis and rotate around the Tm axis by ∼ 20° upon Ca2+ binding. |
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Keywords: | Tn troponin Tm tropomyosin FRET fluorescence resonance energy transfer S1 myosin subfragment-1 TnT25k TnT 25-kDa fragment DABMI 4-dimethyl-aminophenylazophenyl 4&prime -maleimide IAEDANS 5-(2-iodoacetylaminoethyl) aminonaphthalene 1-sulfonic acid AEDANS IAEDANS attached to cysteine residue |
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