Tropomyosin Flexibility Evaluated by Electron Microscopy Image Analysis

Abstract

Movement of tropomyosin (Tm) on thin filaments in response to Ca2+-binding to troponin and myosin binding to actin is an inherent feature of muscle regulation. As part of this process, the cable-like mechanical properties of the Tm coiled-coil are thought to underlie cooperative on and off switching of contraction. In principle, movement of Tm over the flat surface of actin may not require significant molecular flexibility. However, local perturbations caused, for example, by myosin binding on actin may necessitate some plasticity of the Tm molecule. In contrast, any large-scale Tm flexibility might dampen its cooperative movement. In the current study, we directly assessed the flexibility of Tm by examining EM images of both rotary shadowed and negatively stained molecules. Single Tm molecules in both image sets showed no obvious signs of sharp bending or kinks, and displayed contours close to those predicted from a high-resolution 3D model of Tm (Lorenz et al., 1995). Short multimeric strings of end-to-end bonded Tm were commonly observed in the rotary shadowed images. These showed no pronounced bending or joints at the intermolecular junctions. The persistence length of Tm was calculated to be over twice the length of the molecule based on these EM images. The data as a whole imply that Tm molecules exhibit an intrinsic stiffness sufficient to contribute to cooperativity on thin filaments and thus are consistent with models of muscle regulation (e.g. Lehrer & Geeves, 1998; Lehman et al., 2000). A tropomyosin mutant in which the coiled-coil interface was destabilized by introduction of Ala clusters in the 2nd and 3rd periodic repeats (Y60A-L64A-L106A; Singh & Hitchcock-DeGregori, unpublished) was also examined by EM. No obvious extra bending was observed, although the calculated persistence length was significantly shortened, showing the sensitivity of the technique.