title: A three-dimensional FRET analysis to construct an atomic model of the actin-tropomyosin complex on a reconstituted thin filament.
abstract: Fluorescence resonance energy transfer (FRET) was used to construct an atomic model of the actin-tropomyosin (Tm) complex on a reconstituted thin filament. We generated five single-cysteine mutants in the 146-174 region of rabbit skeletal muscle α-Tm. An energy donor probe was attached to a single-cysteine Tm residue, while an energy acceptor probe was located in 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 and Tm, we searched all possible arrangements for Tm segment 146-174 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 the Tm segment on the F-actin filament. Furthermore, we generated a set of five single-cysteine mutants in each of the four Tm regions 41-69, 83-111, 216-244, and 252-279. Using the same procedures, we determined each segment's location on the F-actin filament. In the best-fit model, Tm runs along actin residues 217-236, which were reported to compose the Tm binding site. Electrostatic, hydrogen-bonding, and hydrophobic interactions are involved in actin and Tm binding. The C-terminal region of Tm was observed to contact actin more closely than did the N-terminal region. Tm contacts more residues on actin without Ca(2+) than with it. Ca(2+)-induced changes on the actin-Tm contact surface strongly affect the F-actin structure, which is important for muscle regulation.
authors: Division of Applied Chemistry and Biotechnology, Graduate School of Engineering Science, University of Fukui, Fukui 910-8507, Japan. mmiki@u-fukui.ac.jp
authors: Masao
authors: M
authors: Miki
authors: Satoshi
authors: S
authors: Makimura
authors: Takahiro
authors: T
authors: Saitoh
authors: Masashi
authors: M
authors: Bunya
authors: Yasuyuki
authors: Y
authors: Sugahara
authors: Yutaka
authors: Y
authors: Ueno
authors: Chieko
authors: C
authors: Kimura-Sakiyama
authors: Hidetaka
authors: H
authors: Tobita
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry; D000199:Actins / Q000737:chemistry*; D000818:Animals; D002118:Calcium / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D009154:Mutation; D011485:Protein Binding; D011817:Rabbits; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics
chemical_list: D000199:Actins; D014335:Tropomyosin; D002118:Calcium