Biochemical and Biological Properties of Myosins

Heart, Lung, And Blood Institute

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Abstract

We had found previously that chimeras in which the cardiomyopathy (CM) loop of Dictyostelium myosin II was replaced by the CM loop of either smooth muscle or cardiac muscle myosin II were biochemically impaired in vitro and functionally impaired in vivo. Also, the single point mutation of Ala400 to Val was almost completely inactive both biochemically and biologically while the reverse mutation, Val400Ala, in the cardiac chimera restored almost complete activity to this inactive chimera despite the fact that it still differed in 7 of the 11 residues in the CM-loop. Transient kinetic analysis of the ATPase cycle catalyzed by acto-S1 showed that the affected step in all of the chimeras was the release of P1 from the acto-S1.ADP.Pi intermediate. The presence of Val at position 400 also greatly reduced the affinity of S1 for F-actin in the absence of nucleotide. These results indicate that the conformation of the CM-loop affects the overall conformation of the motor domain affecting the overall complementarity between actin and myosin. The tail of Acanthamoeba myosin IC contain a basic region (BR) followed by a glycine-proline-alainine rich region (GPA1), and SH3 region and a GPA2 region. Previous results from cryoelectron microscopy indicated that the tail was folded back on itself after the BR with GPA1 and GPA2 interacting with BR. Preliminary analysis of NMR data of N15-, and N15/C13-labeled full length tail and the separately expressed N-terminal and C-terminal halves of the tail are consistent with this interpretations there are significant differences in chemical shifts and intensities of backbone amide atoms in the full-length tail compared to the separate halves. Acanthamoeba myosin II is regulated by phosphorylation of three serines residues in the C-terminal non-helical tailpiece. We are trying to understand the mechanism by which this occurs given that the regulatory serine residues are separated from the ATPase site they regulated by about 90 nm of coiled-coil helix. Replacing the serines with either aspartate or glutamate had no effect on the biophysical properties of tail constructs. cDNAs for the heavy chain and two light chains were co-expressed in Sf9 cells with good yield of fully active, regulatable myosin II. Expressed enzyme was fully active when the regulatory serines were replaced by either aspartates or glutamates, indicating that neither Asp nor Glu mimics phosphoSer in this situation.