Functional homodimers and heterodimers of recombinant smooth muscle tropomyosin

Abstract

Skeletal and smooth muscle tropomyosin (Tm) require acetylation of their N-termini to bind strongly to actin. Tm containing an N-terminal alanine-serine (AS) extension to mimic acetylation has been widely used to increase binding. The current study investigates the ability of an N-terminal AS extension to mimic native acetylation for both alpha alpha and beta beta smooth Tm homodimers. We show that (1) AS alpha-Tm binds actin 100-fold tighter than alpha-Tm and 2-fold tighter than native smooth alphabeta-Tm, (2) beta-Tm requires an AS extension to bind actin, and (3) AS beta-Tm binds actin 10-fold weaker than AS alpha-Tm. Tm is present in smooth muscle tissues as >95% heterodimer; therefore, we studied the binding of recombinant alphabeta heterodimers with different AS extensions. This study shows that recombinant Tm requires an AS extension on both alpha and beta chains to bind like native Tm and that the alpha chain contributes more to actin binding than the beta chain. Once assembled onto an actin filament, all smooth muscle Tm's regulate S1 binding to actin Tm in the same way, irrespective of the presence of an AS extension.

Figure 1. Sedimentation assay of tropomyosin binding to actin

A) 10 μM actin and different β-Tm concentrations (0.5 – 7.0 μM) were spun at 100000 g and the supernatants (upper gel) and pellets (lower gel) analysed by SDS-PAGE. B) Analysis of the fraction of β-Tm binding to actin from the relative band densities Binding constants (K_50%).the fraction of Tm bound to actin was estimated from the density of the bands in the pellet and plotted against the free Tm concentration. The fitted line is the least squares best-fit to the Hill Equation. Conditions: 100 mM KCl, 5 mM MgCl₂, 20 mM MOPS, pH 7.0, 20° C.

Figure 2

Fluorescence titration of pyrene actin by S1 in 50 nM pyrene actin plus 2 μM native (dots) and ASα- ASβTm (dashes) titrated with S1 between 0 and 250 nM. The data was fitted to the 2-state cooperative binding model with the fits superimposed as a solid line on the raw data. Fitted parameters are given in Table 1. Conditions: 100 mM KCl, 5 mM MgCl₂, 20 mM MOPS, pH 7.0, 20° C.

Figure 3

Verification of formation of αβ heterodimers by gel electrophoresis after cross-linking treatment SDS-PAGE of crosslinked Tm dimers run under oxidising conditions. Lane 1, αα homodimer alone; lane 2, a 1:1 mixture of αα homodimer with αβ heterodimer; lane 3, αβ heterodimer; lane 4, ββ homodimer; lane 5, a 1:1 mixture of ββ homodimer with αβ heterodimer; lane 5, αβ heterodimer. All samples were denatured in 20mM DTT at 60°C, left to cool to 37°C before cross-linking at 20°C via a Cu²⁺catalysed K₃Fe(CN)₆ reaction.

Figure 4. Affinities of ASα-Tm/ASβ-Tm (open circles) compared with Native Gizzard Tm (squares) for actin as determined by cosedimentation analysis

Conditions are the same as described in figure 1.