Chimeric regulatory light chains as probes of smooth muscle myosin function.
Functional domains of the smooth muscle regulatory light chain (LC) were identified from the assembly and motor properties of smooth muscle myosin containing chimeric LCs, in which the N- and C-terminal halves of the smooth and skeletal LCs were interchanged. A C-terminal domain was also expressed. The affinity of these LCs for the smooth muscle myosin heavy chain (HC) is: wild-type LC > N-skeletal/C-smooth > N-smooth/C-skeletal approximately skeletal LC >> C-terminal domain. The C-terminal half of the LC thus contains an isoform-specific HC binding site, but the two halves of the LC must interact for tight binding. Smooth muscle myosin containing chimeric or skeletal LCs can no longer assume the folded monomeric conformation, suggesting that control of assembly involves both halves of the LC. Dephosphorylation/phosphorylation of the N-skeletal/C-smooth chimera nonetheless regulates the ability of smooth muscle myosin to move actin. Myosin containing phosphorylated N-smooth/C-skeletal or skeletal LCs, in contrast, is locked in the "off" state. Interactions between the stronger binding C-terminal domain of the LC and the HC are therefore primarily responsible for the regulatory capabilities of this subunit. Localization of the regulatory LC at the head/rod junction by electron microscopy establishes that phosphorylation-induced changes must be transmitted over 10 nm within the head for product release to be enhanced.
Trybus
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Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254.
Chatman
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D000595:Amino Acid Sequence; D000818:Animals; D015335:Molecular Probes / Q000737:chemistry; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000502:physiology*; D009154:Mutation; D009218:Myosins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014422:Turkeys
D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; D009218:Myosins