BACKGROUND Fission yeast possesses three unconventional myosins: Myo1p (a class I myosin that functions at endocytic actin patches) and Myo51p and Myo52p (class V myosins that function at contractile rings and actin cables, respectively). Here we used a combination of in vivo and in vitro approaches to investigate how changes in the actin track influence the motor activity and spatial regulatio...

Regulation of muscle contraction is a very cooperative process. The presence of tropomyosin on the thin filament is both necessary and sufficient for cooperativity to occur. Data recently obtained with various tropomyosin isoforms and mutants help us to understand better the structural requirements in the thin filament for cooperative protein interactions. Forming an end-to-end overlap between ...

Regulation of myosin and filamentous actin interaction by tropomyosin is a central feature of contractile events in muscle and nonmuscle cells. However, little is known about molecular interactions within the complex and the trajectory of tropomyosin movement between its "open" and "closed" positions on the actin filament. Here, we report the 8 Å resolution structure of the rigor (nucleotide-fr...

Cardiomyopathies are a major health problem, with inherited cardiomyopathies, many of which are caused by mutations in genes encoding sarcomeric proteins, constituting an ever-increasing fraction of cases. To begin to study the mechanisms by which these mutations cause disease, we have employed an integrative modelling approach to study the interactions between tropomyosin and actin. Starting f...

The interactions of vascular smooth muscle caldesmon with actin, tropomyosin, and calmodulin were determined under conditions in which the four proteins can form reconstituted Ca2+-sensitive smooth muscle thin filaments. Caldesmon bound to actin in a complex fashion with high affinity sites (K = 10(7) M-1) saturating at a stoichiometry of 1 per 28 actins, and lower affinity sites at 1 per 7 act...

Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation incre...

We compared the mechanisms by which caldesmon inhibits actin and actin-tropomyosin activation of myosin subfragment 1 (S1) MgATPase activity. Caldesmon always inhibited actin activation by displacing S1.ADP.Pi from actin and inhibition required at least 0.7 caldesmon molecules bond per actin for 90% inhibition. Caldesmon inhibited actin-tropomyosin without any displacement of S1.ADP.Pi; thus it...

The binding of tropomyosin to F-actin is strongly dependent on M&+ concentration. With muscle actin, in the presence of 2 mu ATP, binding begins at 4 mM M&+ and is complete at about 4.75 mu Mg+ while, with Acanthamoeba actin, binding is initiated at 6 mu M&+ and reaches saturation at 8.5 InM Mg+. Copolymers of muscle and Acanthamoeba actin, however, behave as unique species of actin, each with ...

We showed previously that activated Ras, but not Raf, causes transformation of RIE-1 epithelial cells, demonstrating the importance of Raf-independent pathways in mediating Ras transformation. To assess the mechanism by which Raf-independent effector signaling pathways contribute to Ras-mediated transformation, we recently utilized representational difference analysis to identify genes expresse...

By means of new or modified procedures troponin-tropomyosin complex was fractionated into three components plus tropomyosin, and then reconstituted in the absence of urea. Tropomyosin was separated from troponin by hydroxyapatite column chromatography, a method giving sharper separation than the previously used technique of isoelectric precipitation. The chromatography also separated the tropom...