A three-dimensional FRET analysis to construct an atomic model of the actin-tropomyosin-troponin core domain complex on a muscle thin filament.

Masao Miki, Satoshi Makimura, Yasuyuki Sugahara, Ryuta Yamada, Masashi Bunya, Takahiro Saitoh, Hidetaka Tobita

Division of Applied Chemistry and Biotechnology, Graduate School of Engineering Science, University of Fukui, Fukui, Japan. mmiki@u-fukui.ac.jp

Journal of molecular biology 2012 Jun 29

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It is essential to know the detailed structure of the thin filament to understand the regulation mechanism of striated muscle contraction. Fluorescence resonance energy transfer (FRET) was used to construct an atomic model of the actin-tropomyosin (Tm)-troponin (Tn) core domain complex. We generated single-cysteine mutants in the 167-195 region of Tm and in TnC, TnI, and the β-TnT 25-kDa fragment, and each was attached with an energy donor probe. An energy acceptor probe was located at actin Gln41, actin Cys374, or the actin nucleotide-binding site. From these donor-acceptor pairs, FRET efficiencies were determined with and without Ca(2+). Using the atomic coordinates for F-actin, Tm, and the Tn core domain, we searched all possible arrangements for Tm or the Tn core domain on F-actin to calculate the FRET efficiency for each donor-acceptor pair in each arrangement. By minimizing the squared sum of deviations for the calculated FRET efficiencies from the observed FRET efficiencies, we determined the location of Tm segment 167-195 and the Tn core domain on F-actin with and without Ca(2+). The bulk of the Tn core domain is located near actin subdomains 3 and 4. The central helix of TnC is nearly perpendicular to the F-actin axis, directing the N-terminal domain of TnC toward the actin outer domain. The C-terminal region in the I-T arm forms a four-helix-bundle structure with the Tm 175-185 region. After Ca(2+) release, the Tn core domain moves toward the actin outer domain and closer to the center of the F-actin axis. Copyright © 2012 Elsevier Ltd. All rights reserved.

Citation

Masao Miki, Satoshi Makimura, Yasuyuki Sugahara, Ryuta Yamada, Masashi Bunya, Takahiro Saitoh, Hidetaka Tobita. A three-dimensional FRET analysis to construct an atomic model of the actin-tropomyosin-troponin core domain complex on a muscle thin filament. Journal of molecular biology. 2012 Jun 29;420(1-2):40-55