Visualization of Troponin on Muscle Thin Filaments by Single Particle Analysis

Abstract

Troponin on the thin filaments of striated muscle couples Ca-concentration changes to movement of tropomyosin. Ca-free troponin is thought to shift tropomyosin to the myosin-blocking position, a constraint that is released after Ca binds. Although the location and regulatory movements of tropomyosin have been defined at near-atomic resolution, the organization of troponin on thin filaments has not been determined definitively, and different models of troponin position on actin are contradictory. Here novel single-particle analysis (SPA) protocols were designed to reconstruct thin filament structure from electron micrographs of negatively-stained cardiac muscle thin filaments at low-Ca. Troponin-tropomyosin has a tendency to dissociate from filaments under EM conditions, and only filaments showing evidence of bound troponin were chosen for analysis. The axial location of troponin densities on actin was identified by newly developed algorithms. Filament polarity was determined according to Narita and Maeda (2007). Filaments were divided computationally into 46.8 nm particles centered on troponin. SPA was carried out on these particles without applying helical averaging, and successively refined reconstructions did not degrade the troponin-tropomyosin signal. Tropomyosin strands extended smoothly and continuously over adjacent actin monomers along the long-pitch filament helix, occupying the myosin blocking position. Tropomyosin atomic models fitted well to corresponding densities close to the binding site on actin described in Li et al. (2011) for troponin-free filaments. The troponin core-domain position and orientation deviated slightly from those proposed by Pirani et al. (2006). A broadened tropomyosin strand density found on the barbed-end side of the troponin core-domain appeared to bifurcate to link tropomyosin and the core-domain. We attribute this density to the tail of troponin-T, whose location corroborates the polarity of troponin on actin proposed by Flicker et al. (1982).