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.

A Three-Dimensional FRET Analysis to Construct an Atomic Model of the Actin–Tropomyosin–Troponin Core Domain Complex on a Muscle Thin Filament

“…IAEDANS was also attached to each cysteine in the singlecysteine Tm mutants at positions 167, 174, 181, 188, and 195. We measured Ca 2+ regulation of actin-activated S1-ATPase activity for the labeled Tm mutants as we have previously reported, 19 and the relative Ca 2 + sensitivity of each AEDANSlabeled Tm mutant is shown in Table 2. The energy acceptor molecules 4-dimethyl-aminophenylazophenyl 4′-maleimide (DABMI) and fluorescein cadaverine (FLC) were attached to actin Cys374 and Gln41, respectively, and the ADP bound to F-actin was replaced with 2′(or 3′)-O-(2,4,6-trinitrophenyl) (TNP)-ADP.…”

“…The FRET measurements using different acceptor labeling ratios provide information on the radial coordinates of the donor and acceptor. Therefore, to increase the accuracy in locating the Tn core domain on F-actin, we measured the transfer efficiencies at different acceptor labeling ratios (two) as described by Miki et al 19 The E obs values are summarized in Table 1.…”

“…The five nearest-neighbor distances were used to calculate the FRET efficiency, as previously analyzed for locating Tm on F-actin. 19 Using these distance values, we calculated the energy transfer efficiency (E calc ) for each FRET pair In the leftmost column, the percentage denoted beside the Tn residue number shows relative Ca 2+ sensitivity (100% for native Tn). Q 0 , the quantum yield of AEDANS attached to the Tn subunit on the reconstituted thin filament in the presence of Ca 2+ (+Ca) and in the absence of Ca 2+ (−Ca); R 0 , Förster's critical distance (in angstroms) in the presence of Ca 2+ (+Ca) and in the absence of Ca 2+ (− Ca); A/P, acceptor/ protein (actin) labeling ratio; E obs , measured energy transfer efficiency (errors in E obs were within ± 0.03); E calc (A) and E calc (B), calculated energy transfer efficiency at two best-fit models, A-model and B-model, respectively.…”

“…18 By extending this method, we constructed an atomic model of the actin-Tm complex on a reconstituted thin filament with and without Ca 2+ . 19 The atomic coordinates of Lorenz et al's F-actin model (the Lorenz model) 8 and the pig skeletal Tm crystal structure (Protein Data Bank code: 1C1G) 4 were used. The model shows which actin and Tm residues are involved in the interaction that includes electrostatic, hydrogen-bonding, and hydrophobic forces.…”

“…By systematically translating and rotating the Tn core domain around the F-actin filament, we obtained the best-fit models with and without Ca 2+ by minimizing the squared sum of deviations of the calculated energy transfer efficiencies from the observed values for all of the FRET measurements at each orientation. Furthermore, we determined the location of Tm segment 167-195 on the F-actin filament, using the same procedures as previously reported 19 except that we used the coordinates from the Fujii model instead of the Lorenz model. Next, we constructed an atomic model of the Tn core domain-Tm (167-195)-F-actin complex with and without Ca 2+ .…”