Myosin-induced movement of alphaalpha, alphabeta, and betabeta smooth muscle tropomyosin on actin observed by multisite FRET

Abstract

The interaction of the alphaalpha, betabeta, and alphabeta smooth muscle tropomyosin (Tm) isoforms with F-actin was systematically studied in the absence and in the presence of myosin subfragment 1 (S1) using multifrequency phase/modulation Förster resonance energy transfer (FRET). A Gaussian double distance distribution model was adopted to fit FRET data between a 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid donor at either Cys-36 of the beta-chain or Cys-190 of the alpha-chain and a 4-dimethylaminophenylazophenyl 4'-maleimide acceptor at Cys-374 of F-actin. Experimental data were obtained for singly and doubly labeled alphabeta Tm (donor only at alpha, only at beta, or both) and for doubly labeled alphaalpha or betabeta Tm. Data for singly labeled alphabetaTm were combined in a global analysis with doubly labeled alphabetaTm. In all doubly labeled isoforms, upon S1 binding, one donor-acceptor "apparent" distance increased slightly by 0.5-2 A, whereas the other decreased by 6-9 A. These changes are consistent with a uniform "rolling" motion of Tm over the F-actin surface. The analysis indicates that Tm occupies relatively well-defined positions, with some flexibility, in both the predominantly closed (-S1) and open (+S1) thin-filament states. The results for the alphabetaTm heterodimer indicate that the local twofold symmetry of alphaalpha or betabeta Tm is effectively broken in alphabetaTm bound to F-actin, which implies a difference between the alpha- and beta-chains in terms of their interaction with F-actin.

FIGURE 1

Atomic coordinate model of the F-actin·Tm complex from Lorenz et al. (1993, 1995). The model is formed by six actin monomers (wire frame) and two αβTm molecules (α-chain, cyan ribbon; β-chain, blue ribbon) on opposing sides of the F-actin helix. The donor and acceptor moieties (space-fill) are AEDANS attached to βTm Cys-36 (D₁) and DABMI attached to F-actin Cys-374 (A₁–A₆), respectively. Displayed using the program RasMol (Bernstein, 1999).

FIGURE 2

Frequency-domain phase and modulation data and fits for AEDANS-labeled Tm alone and in complex with F-actin and S1. The phase angle increases and the modulation decreases with increasing frequency. (a) Tm alone (all combinations of α- and β-chains, singly and doubly labeled are shown). (b) Doubly labeled Tm heterodimer (α^*β^* Tm) + F-actin in the absence (□) and in the presence (+) of S1. The Tm-alone fit (thicker line) is shown for comparison. (c) Phase (δ_φ) and modulation (δ_m) residuals for a typical fit. The parameters recovered from the fits are reported in Tables 1–3.

FIGURE 3

(a) Frequency-domain phase and modulation data in the presence of energy transfer and fits for α^*β^* AEDANS donor-labeled Tm in complex with DABMI acceptor-labeled F-actin in the absence (□) and in the presence (+) of S1. The fit of the corresponding donor-only F-actin·Tm complex (D-only, thicker line) is shown for comparison. (b) Phase (δ_φ) and modulation (δ_m) residuals for a typical fit. The parameters recovered from the fits are reported in Tables 4 and 5.

FIGURE 4

S1-induced azimuthal and axial movement of the αβTm heterodimer corresponding to the atomic coordinate-distance distribution analysis reported in Table 7. The longitudinal view of the thin filament along theF-actin axis (C) shows two adjacent actin monomers (green and red wire frame); the ±S1 fitted positions of the Tm region around the donor at Cys-190 (Glu-187–Leu-193, blue and cyan ribbons; Cys-190, space-fill) and at Cys-36 (Glu-33–Leu-39, blue and cyan ribbons; Cys-36, space-fill); and the fitted average position of the DABMI moiety (space-fill; see text for details). The fitted Tm movement is a combination of an azimuthal (around the F-actin axis) and an axial (around its own interchain axis) rotation. Displayed using the program RasMol (Bernstein, 1999).

FIGURE 5

Comparison between the position of the region around the donor at Cys-190 of the α-chain and the position of the region around the donor at Cys-36 of the β-chain in the αβTm heterodimer in presence of S1, corresponding to the atomic coordinate-distance distribution analysis reported in Table 7 (+S1). The longitudinal view of the thin filament along the F-actin axis (C) is similar to Fig. 4 and shows two adjacent actin monomers (green and red wire frame); the +S1 fitted positions of the Tm region around the donor at Cys-190 (Glu-187–Leu-193, blue and cyan space-fill; Cys-190 and AEDANS, space-fill) and at Cys-36 (Glu-33–Leu-39, blue and cyan space-fill; Cys-36 and AEDANS, space-fill); and the fitted average position of the DABMI moiety (space-fill; see text for details). Displayed using the program RasMol (Bernstein, 1999).

FIGURE 6

Comparison between best-fit average position of the DABMI moiety attached to F-actin Cys-374 (left) and the position of a TMR moiety linked to Cys-374 of G-actin (same backbone orientation as F-actin is shown), determined by x-ray crystallography (Otterbein et al., 2001) (right). The view, along the F-actin axis, shows one actin monomer (red wire frame), a ribbon rendering of the F-actin C-terminal region (from Gly-366 to Cys-374, left, or to Arg-372, right) and the attached moieties (DABMI, left, and TMR, right, space-fill). Displayed using the program RasMol (Bernstein, 1999).