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Direct observation of tropomyosin binding to actin filaments.

ABSTRACT: Tropomyosin is an elongated ?-helical coiled coil that binds to seven consecutive actin subunits along the long-pitch helix of actin filaments. Once bound, tropomyosin polymerizes end-to-end and both stabilizes F-actin and regulates access of various actin-binding proteins including myosin in muscle and nonmuscle cells. Single tropomyosin molecules bind weakly to F-actin with millimolar Kd , whereas the end-to-end linked tropomyosin associates with about a 1000-fold greater affinity. Despite years of study, the assembly mechanism of tropomyosin onto actin filaments remains unclear. In this study, we used total internal reflection fluorescence microscopy to directly monitor the cooperative binding of fluorescently labeled tropomyosin molecules to phalloidin-stabilized actin filaments. We find that tropomyosin molecules assemble from multiple growth sites after random low-affinity binding of single molecules to actin. As the length of the tropomyosin chain increases, the probability of detachment decreases, which leads to further chain growth. Tropomyosin chain extension is linearly dependent on the concentration of tropomyosin, occurring at approximately 100 monomers/(?M*s). The random tropomyosin binding to F-actin leads to discontinuous end-to-end association where gaps in the chain continuity smaller than the required seven sequential actin monomers are available. Direct observation of tropomyosin detachment revealed the number of gaps in actin-bound tropomyosin, the time course of gap annealing, and the eventual filament saturation process.

SUBMITTER: Schmidt WM

PROVIDER: S-EPMC4529784 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Direct observation of tropomyosin binding to actin filaments.

Schmidt William M WM Lehman William W Moore Jeffrey R JR

Cytoskeleton (Hoboken, N.J.) 20150630 6

Tropomyosin is an elongated α-helical coiled coil that binds to seven consecutive actin subunits along the long-pitch helix of actin filaments. Once bound, tropomyosin polymerizes end-to-end and both stabilizes F-actin and regulates access of various actin-binding proteins including myosin in muscle and nonmuscle cells. Single tropomyosin molecules bind weakly to F-actin with millimolar Kd , whereas the end-to-end linked tropomyosin associates with about a 1000-fold greater affinity. Despite yea ...[more]

PMID: 26033920

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Project description:Experimental approaches such as fiber diffraction and cryo-electron microscopy reconstruction have defined regulatory positions of tropomyosin on actin but have not, as yet, succeeded at determining key atomic-level contacts between these proteins or fully substantiated the dynamics of their interactions at a structural level. To overcome this deficiency, we have previously employed computational approaches to deduce global dynamics of thin filament components by energy landscape determination and molecular dynamics simulations. Still, these approaches remain computationally challenging for any complex and large macromolecular assembly like the thin filament. For example, tropomyosin cable wrapping around actin of thin filaments features both head-to-tail polymeric interactions and local twisting, both of which depart from strict superhelical symmetry. This produces a complex energy surface that is difficult to model and thus to evaluate globally. Therefore, at this stage of our understanding, assessing global molecular dynamics can prove to be inherently impractical. As an alternative, we adopted a "divide and conquer" protocol to investigate actin-tropomyosin interactions at an atomistic level. Here, we first employed unbiased protein-protein docking tools to identify binding specificity of individual tropomyosin pseudorepeat segments over the actin surface. Accordingly, tropomyosin "ligand" segments were rotated and translated over potential "target" binding sites on F-actin where the corresponding interaction energetics of billions of conformational poses were ranked by the programs PIPER and ClusPro. These data were used to assess favorable interactions and then to rebuild models of seamless and continuous tropomyosin cables over the F-actin substrate, which were optimized further by flexible fitting routines and molecular dynamics. The models generated azimuthally distinct regulatory positions for tropomyosin cables along thin filaments on actin dominated by stereo-specific head-to-tail overlap linkage. The outcomes are in good agreement with current cryo-electron microscopy topology and consistent with long-thought residue-to-residue interactions between actin and tropomyosin.

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Direct observation of tropomyosin binding to actin filaments.

Publications

Direct observation of tropomyosin binding to actin filaments.

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