Acanthamoeba myosin II rod is a long alpha-helical dimeric coiled-coil with a flexible hinge containing a helix-breaking proline. Previously, the thermal stabilities of the complete 'wild-type' rod domain of myosin II (residues 849-1509) and hinge mutants in 0.6 and 2.2 M KCl, pH 7.5, have been determined. Analytical ultracentrifugation also has been used to characterize the size and shape (frictional ratio) of myosin II wild-type and hinge mutant rods. Sedimentation properties of minifilaments formed by dialysis of the wild-type and point and deletion mutants vs. low ionic strength buffer with 5 mM Mg (II) at pH 7.5 also have been determined. In addition, the flexibility of myosin II rod minifilaments as a function of Mg(II) concentration has been measured by electric birefringence; specifically, the Mg(II)-dependent flexible-to-stiff transitions of native myosin II and the wild-type rod minifilaments were found by Rau to be very similar. Mutations of the phosphorylation sites in the C-terminal tail of the myosin II rod have substituted 3 Asp for 3 Ser [S1489D, S1494D, S1499D] (78,615 MW), 3 Ala for 3 Ser [S1489A, S1494A, S1499A] (78,483 MW), and 1 Asp for Ser [S1489D] (78,559 MW) with an amino terminal flag (wild-type rod has 78,531 MW). The wild-type and mutant rods will be used by B. Kunnummal (LCB) to make cofilaments with native myosin II to test if the actin-activated ATPase activity of myosin II heads is inhibited by copolymerized rods containing the Ser-to-Asp substitutions (made to mimic phosphorylation at these sites). If so, the Ser-to-Ala substituted rods in cofilaments with myosin II will act as controls by showing no effect on the head ATPase activity. What we have determined in the Section on Protein Chemistry, LB is that the thermal stabilities of the wild-type rod dimer and the three phosphorylation-site mutant rods are very similar, using equilibrium circular dichroism measurements at 222 nm and a differential scanning calorimeter (VP-DSC) at a slow scan rate of 4.89 C per hour. Sedimentation velocity experiments demonstrated that all rod preparations in 0.6 M KCl at pH 7.5 are dimeric with ellipsoidal axial ratios of about 45.Currently, chimeras of myosin II (with Dictyostelium discoideum heads and collar regions of myosin II joined to either Acanthamoeba myosin II rod or smooth muscle myosin rod) are being examined under monomeric conditions in the absence and presence of nucleotide (AMPPNP) by differential scanning calorimetry (DSC) and in the analytical ultracentrifuge for sedimentation velocity behavior. Reference proteins are intact myosin II from Acanthamoeba and Dictyostelium and intact smooth muscle myosin. DSC results indicate that there are major changes in the thermal stabilities of head domains produced by nucleotide binding and in the coupling and/or cooperativity of head and rod domain unfolding of chimeric proteins. Binding of an ATP analogue appears to produce more compact conformations in the asymmetric, monomeric chimeric myosins II (ca. 6 S), as measured by difference sedimentation velocity.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Intramural Research (Z01)
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U.S. National Heart Lung and Blood Inst
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Liu, X; Shu, S; Yamashita, R A et al. (2000) Chimeras of Dictyostelium myosin II head and neck domains with Acanthamoeba or chicken smooth muscle myosin II tail domain have greatly increased and unregulated actin-dependent MgATPase activity. Proc Natl Acad Sci U S A 97:12553-8
Korn, E D (2000) Coevolution of head, neck, and tail domains of myosin heavy chains. Proc Natl Acad Sci U S A 97:12559-64
Parsegian, V A; Rand, R P; Rau, D C (2000) Osmotic stress, crowding, preferential hydration, and binding: A comparison of perspectives. Proc Natl Acad Sci U S A 97:3987-92
Sidorova, N Y; Rau, D C (2000) The dissociation rate of the EcoRI-DNA-specific complex is linked to water activity. Biopolymers 53:363-8
Rand, R P; Parsegian, V A; Rau, D C (2000) Intracellular osmotic action. Cell Mol Life Sci 57:1018-32
Sidorova, N Y; Rau, D C (1999) Removing water from an EcoRI-noncognate DNA complex with osmotic stress. J Biomol Struct Dyn 17:19-31
Redowicz, M J; Hammer 3rd, J A; Bowers, B et al. (1999) Flexibility of Acanthamoeba myosin rod minifilaments. Biochemistry 38:7243-52