Flexibility of Acanthamoeba Myosin Rod Minifilaments

Abstract

Previous electric birefringence experiments have shown that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II correlates with the ability of minifilaments to cycle between flexible and stiff conformations. The cooperative transition between conformations was shown to depend on Mg2+ concentration, on ATP binding, and on the state of phosphorylation of three serines in the C-terminal end of the heavy chains. Since the junction between the heavy meromyosin (HMM) and light meromyosin (LMM) regions is expected to disrupt the alpha-helical coiled-coil structure of the rod, this region was anticipated to be the flexible site. We have now cloned and expressed the wild-type rod (residues 849-1509 of the full-length heavy chain) and rods mutated within the junction in order to test this. The sedimentation and electric birefringence properties of minifilaments formed by rods and by native myosin II are strikingly similar. In particular, the Mg2+-dependent flexible-to-stiff transitions of native myosin II and wild-type rod minifilaments are virtually superimposable. Mutations within the junction between the HMM and LMM regions of the rod modulate the ability of Mg2+ to stabilize the stiff conformation. Less Mg2+ is required to induce minifilament stiffening if proline-1244 is replaced with alanine. Deleting the entire junction region (25 amino acids) results in a even greater decrease in the Mg2+ concentration necessary for the transition. The HMM-LMM junction does indeed seem to act as a Mg2+-dependent flexible hinge.

Independent Movement, Dimerization and Stability of Tandem Repeats of Chicken Brain α-Spectrin

“…46,47 In fact, if unfolding of spectrin fragments by atomic force microscopy (AFM) is a paradigm of some of the events of red cell deformation in vivo, the occurrence of single and two-repeat unfolding events observed in that regime 36 suggests that cooperative unfolding is essential to spectrin function. As shown previously and here, the cooperativity characteristic of the heat-induced 25,45 and urea-induced 25,44 unfolding of tandem spectrin repeats differs from that of the unfolding of a single repeat, insofar as two repeats can unfold as a single unit with unique thermodynamic parameters, generally indicating greater stability.…”

Computational simulations reveal that Abl activity controls cohesiveness of actin networks in growth cones

“…, 1993 ). Myosin and α-actinin molecules are represented as Hookean springs with equilibrium lengths based on experimental measurements ( Redowicz et al. , 1999 ; Ferrer et al.…”

“…Bending deformations around hinge points are allowed corresponding to a persistence length of 16.7µm. (Ott et al, 1993) Myosin and α-actinin molecules are represented as Hookean springs with equilibrium lengths based on experimental measurements (Redowicz et al, 1999;Ferrer et al, 2008). As a result, chemical reactions such as crosslinker (un)binding, motor (un)binding, and motor walking events are defined between filaments that satisfy the appropriate distance thresholds.…”