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Collins, Agnieszka (1)
Fischer, Stefan (1)
Huang, Renjian (1)
Jensen, Mikkel Herholdt (1)
Li, Xiaochuan (1)
Moore, Jeffrey R (1)
Moore, Jeffrey R. (1)
Nirody, Jasmine (1)
Wang, Chih-Lueh Albert (1)
Year of Publication
Structural implications of conserved aspartate residues located in tropomyosin’s coiled-coil core
Moore, Jeffrey R.
Polar residues lying between adjacent α-helical chains of coiled-coils often contribute to coiled-coil curvature and flexibility, while more typical core hydrophobic residues anneal the chains together. In tropomyosins, ranging from smooth and skeletal muscle to cytoplasmic isoforms, a highly conserved Asp at residue 137 places negative charges within the tropomyosin coiled-coil core in a position which may affect the conformation needed for tropomyosin binding and regulatory movements on actin. Proteolytic susceptibility suggested that substituting a canonical Leu for the naturally occurring Asp at residue 137 increases inter-chain rigidity by stabilizing the tropomyosin coiled-coil. Using molecular dynamics, we now directly assess changes in coiled-coil curvature and flexibility caused by such mutants. Although the coiled-coil flexibility is modestly diminished near the residue 137 mutation site, as expected, a delocalized increase in flexibility along the overall coiled-coil is observed. Even though the average shape of the D137L tropomyosin is straighter than that of wild-type tropomyosin, it is still capable of binding actin due to this increase in flexibility. We conclude that the conserved, non-canonical Asp-137 destabilizes the local structure resulting in a local flexible region in the middle of tropomyosin that normally is important for tropomyosin steady-state equilibrium position on actin.
Coiled-coil; Flexibility; Molecular Dynamics; heptad repeat; tropomyosin
Structural studies on maturing actin filaments
Jensen, Mikkel Herholdt
Moore, Jeffrey R
Wang, Chih-Lueh Albert
We have previously reported that actin undergoes a conformational transition (which we named “maturation”) during polymerization, and that the actin-binding protein, caldesmon (CaD), when added at an early phase of polymerization, interferes with this process (Huang et al. J Biol Chem 2010; 285:71). The pre-transition filament is characterized by relatively low pyrene-fluorescence intensity when pyrene-labeled actin is used as a reporter of subunit assembly into filaments, whereas the mature filament emits a characteristic enhanced fluorescence. Previously reported co-sedimentation experiments suggest that filament formation is not inhibited by the presence of CaD, despite blocking the transition associated with filament maturation. In this study we visualized structural effects of CaD on the assembly of actin filaments by TIRF and electron microscopy. CaD-free actin forms “rough” filaments with irregular edges and indistinct subunit organization during the initial phase (∼20 min under our conditions) of polymerization as reported previously by others (Steinmetz et al. J Cell Biol 1997; 138:559; Galinska-Rakoczy et al. J Mol Biol 2009; 387:869), which most likely correspond to the pre-transition state preceding the maturation step. Later during the polymerization process “mature” filaments exhibit a smoother F-actin appearance with easily detectible double helically arranged actin subunits. While the inclusion of the actin-binding domain of CaD during actin polymerization does not affect the elongation rate, it is associated with a prolonged pre-transition phase, characterized by a delayed alteration (rough to smooth) of the appearance of filaments, consistent with a later onset of the maturation process.
actin; polymerization; caldesmon; TIRF microscopy; electron microscopy; conformational change; cytoskeleton dynamics
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